Therapeutic compositions

ABSTRACT

This application relates to therapeutic siRNA agents and methods of making and using the agents.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/623,139, filed Jun. 14, 2017, which is a continuation of U.S.application Ser. No. 14/943,612, filed Nov. 17, 2015, which is acontinuation of U.S. application Ser. No. 14/282,769, filed May 20,2014, which is a continuation of U.S. application Ser. No. 13/626,196,filed Sep. 25, 2012, which is a continuation of U.S. application Ser.No. 12/721,413, filed Mar. 10, 2010, which is a continuation of U.S.application Ser. No. 10/548,611, filed Aug. 22, 2006, which is theNational Stage of International Application No. PCT/US2004/007070, filedMar. 8, 2004, which claims the benefit of Application No. 60/452,682,filed Mar. 7, 2003; Application No. 60/462,894, filed Apr. 14, 2003; andApplication No. 60/465,665, filed Apr. 25, 2003; Application No.60/463,772, filed Apr. 17, 2003; Application No. 60/465,802, filed Apr.25, 2003; Application No. 60/493,986, filed Aug. 8, 2003; ApplicationNo. 60/494,597, filed Aug. 11, 2003; Application No. 60/506,341, filedSep. 26, 2003; Application No. 60/518,453, filed Nov. 7, 2003;Application No. 60/454,265, filed Mar. 12, 2003; Application No.60/454,962, filed Mar. 13, 2003; Application No. 60/455,050, filed Mar.13, 2003; Application No. 60/469,612, filed May 9, 2003; Application No.60/510,246, filed Oct. 9, 2003; Application No. 60/510,318, filed Oct.10, 2003. The contents of the above applications are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

The invention relates to RNAi and related methods, e.g., methods ofmaking and using iRNA agents.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811). Short dsRNA directs gene-specific,post-transcriptional silencing in many organisms, including vertebrates,and has provided a new tool for studying gene function. RNAi may involvemRNA degradation.

SUMMARY

A number of advances related to the application of RNAi to the treatmentof subjects are disclosed herein. For example, the invention featuresiRNA agents targeted to specific genes; palindromic iRNA agents; iRNAagents having non canonical monomer pairings; iRNA agents havingparticular structures or architectures e.g., the Z—X—Y or asymmetricaliRNA agents described herein; drug delivery conjugates for the deliveryof iRNA agents; amphipathic substances for the delivery of iRNA agents,as well as iRNA agents having chemical modifications for optimizing aproperty of the iRNA agent. The invention features each of theseadvances broadly as well as in combinations. For example, an iRNA agenttargeted to a specific gene can also include one or more of apalindrome, non canonical, Z—X—Y, or asymmetric structure. Othernonlimiting examples of combinations include an asymmetric structurecombined with a chemical modification, or formulations or methods orroutes of delivery combined with, e.g., chemical modifications orarchitectures described herein. The iRNA agents of the invention caninclude any one of these advances, or pairwise or higher ordercombinations of the separate advances.

In one aspect, the invention features iRNA agents that can target morethan one RNA region, and methods of using and making the iRNA agents.

In another aspect, an iRNA agent includes a first and second sequencethat are sufficiently complementary to each other to hybridize. Thefirst sequence can be complementary to a first target RNA region and thesecond sequence can be complementary to a second target RNA region.

In one embodiment, the first and second sequences of the iRNA agent areon different RNA strands, and the mismatch between the first and secondsequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

In another embodiment, the first and second sequences of the iRNA agentare on the same RNA strand, and in a related embodiment more than 50%,60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent is in bimolecular form.

In another embodiment, the first and second sequences of the iRNA agentare fully complementary to each other.

In one embodiment, the first target RNA region is encoded by a firstgene and the second target RNA region is encoded by a second gene, andin another embodiment, the first and second target RNA regions aredifferent regions of an RNA from a single gene. In another embodiment,the first and second sequences differ by at least 1 and no more than 6nucleotides.

In certain embodiments, the first and second target RNA regions are ontranscripts encoded by first and second sequence variants, e.g., firstand second alleles, of a gene. The sequence variants can be mutations,or polymorphisms, for example.

In certain embodiments, the first target RNA region includes anucleotide substitution, insertion, or deletion relative to the secondtarget RNA region.

In other embodiments, the second target RNA region is a mutant orvariant of the first target RNA region.

In certain embodiments, the first and second target RNA regions compriseviral, e.g., HCV, or human RNA regions. The first and second target RNAregions can also be on variant transcripts of an oncogene or includedifferent mutations of a tumor suppressor gene transcript. In oneembodiment, the oncogene, or tumor suppressor gene is expressed in theliver. In addition, the first and second target RNA regions correspondto hot-spots for genetic variation.

In another aspect, the invention features a mixture of varied iRNA agentmolecules, including one iRNA agent that includes a first sequence and asecond sequence sufficiently complementary to each other to hybridize,and where the first sequence is complementary to a first target RNAregion and the second sequence is complementary to a second target RNAregion. The mixture also includes at least one additional iRNA agentvariety that includes a third sequence and a fourth sequencesufficiently complementary to each other to hybridize, and where thethird sequence is complementary to a third target RNA region and thefourth sequence is complementary to a fourth target RNA region. Inaddition, the first or second sequence is sufficiently complementary tothe third or fourth sequence to be capable of hybridizing to each other.In one embodiment, at least one, two, three or all four of the targetRNA regions are expressed in the liver. Exemplary RNAs are transcribedfrom the apoB-100 gene, glucose-6-phosphatase gene, beta catenin gene,or an HCV gene.

In certain embodiments, the first and second sequences are on the sameor different RNA strands, and the third and fourth sequences are on sameor different RNA strands.

In one embodiment, the mixture further includes a third iRNA agent thatis composed of the first or second sequence and the third or fourthsequence.

In one embodiment, the first sequence is identical to at least one ofthe second, third and fourth sequences, and in another embodiment, thefirst region differs by at least 1 but no more than 6 nucleotides fromat least one of the second, third and fourth regions.

In certain embodiments, the first target RNA region comprises anucleotide substitution, insertion, or deletion relative to the second,third or fourth target RNA region.

The target RNA regions can be variant sequences of a viral or human RNA,and in certain embodiments, at least two of the target RNA regions canbe on variant transcripts of an oncogene or tumor suppressor gene. Inone embodiment, the oncogene or tumor suppressor gene is expressed inthe liver.

In certain embodiments, at least two of the target RNA regionscorrespond to hot-spots for genetic variation.

In one embodiment, the iRNA agents of the invention are formulated forpharmaceutical use. In one aspect, the invention provides a container(e.g., a vial, syringe, nebulizer, etc) to hold the iRNA agentsdescribed herein.

Another aspect of the invention features a method of making an iRNAagent. The method includes constructing an iRNA agent that has a firstsequence complementary to a first target RNA region, and a secondsequence complementary to a second target RNA region. The first andsecond target RNA regions have been identified as being sufficientlycomplementary to each other to be capable of hybridizing. In oneembodiment, the first and second target RNA regions are on transcriptsexpressed in the liver.

In certain embodiments, the first and second target RNA regions cancorrespond to two different regions encoded by one gene, or to regionsencoded by two different genes.

Another aspect of the invention features a method of making an iRNAagent composition. The method includes obtaining or providinginformation about a region of an RNA of a target gene (e.g., a viral orhuman gene, or an oncogene or tumor suppressor, e.g., p53), where theregion has high variability or mutational frequency (e.g., in humans).In addition, information about a plurality of RNA targets within theregion is obtained or provided, where each RNA target corresponds to adifferent variant or mutant of the gene (e.g., a region including thecodon encoding p53 248Q and/or p53 249S). The iRNA agent is constructedsuch that a first sequence is complementary to a first of the pluralityof variant RNA targets (e.g., encoding 249Q) and a second sequence iscomplementary to a second of the plurality of variant RNA targets (e.g.,encoding 249S). The first and second sequences are sufficientlycomplementary to hybridize. In certain embodiments, the target gene canbe a viral or human gene expressed in the liver.

In one embodiment, sequence analysis, e.g., to identify common mutantsin the target gene, is used to identify a region of the target gene thathas high variability or mutational frequency. For example, sequenceanalysis can be used to identify regions of apoB-100 or beta cateninthat have high variability or mutational frequency. In anotherembodiment, the region of the target gene having high variability ormutational frequency is identified by obtaining or providing genotypeinformation about the target gene from a population. In anotherembodiment, the genotype information can be from a population sufferingfrom a liver disorder, such as hepatocellular carcinoma orhepatoblastoma.

Another aspect of the invention features a method of modulatingexpression, e.g., downregulating or silencing, a target gene, byproviding an iRNA agent that has a first sequence and a second sequencesufficiently complementary to each other to hybridize. In addition, thefirst sequence is complementary to a first target RNA region and thesecond sequence is complementary to a second target RNA region.

In one embodiment, the iRNA agent is administered to a subject, e.g., ahuman.

In another embodiment, the first and second sequences are between 15 and30 nucleotides in length.

In one embodiment, the method of modulating expression of the targetgene further includes providing a second iRNA agent that has a thirdsequence complementary to a third target RNA region. The third sequencecan be sufficiently complementary to the first or second sequence to becapable of hybridizing to either the first or second sequence.

Another aspect of the invention features a method of modulatingexpression, e.g., downregulating or silencing, a plurality of targetRNAs, each of the plurality of target RNAs corresponding to a differenttarget gene. The method includes providing an iRNA agent selected byidentifying a first region in a first target RNA of the plurality and asecond region in a second target RNA of the plurality, where the firstand second regions are sufficiently complementary to each other to becapable of hybridizing.

In another aspect of the invention, an iRNA agent molecule includes afirst sequence complementary to a first variant RNA target region and asecond sequence complementary to a second variant RNA target region, andthe first and second variant RNA target regions correspond to first andsecond variants or mutants of a target gene. In certain embodiments, thetarget gene is an apoB-100, beta catenin, or glucose-6 phosphatase gene.

In one embodiment, the target gene is a viral gene (e.g., an HCV gene),tumor suppressor or oncogene.

In another embodiment, the first and second variant target RNA regionsinclude allelic variants of the target gene.

In another embodiment, the first and second variant RNA target regionscomprise mutations (e.g., point mutations) or polymorphisms of thetarget gene.

In one embodiment, the first and second variant RNA target regionscorrespond to hot-spots for genetic variation.

Another aspect of the invention features a plurality (e.g., a panel orbank) of iRNA agents. Each of the iRNA agents of the plurality includesa first sequence complementary to a first variant target RNA region anda second sequence complementary to a second variant target RNA region,where the first and second variant target RNA regions correspond tofirst and second variants of a target gene. In certain embodiments, thevariants are allelic variants of the target gene.

Another aspect of the invention provides a method of identifying an iRNAagent for treating a subject. The method includes providing or obtaininginformation, e.g., a genotype, about a target gene, providing orobtaining information about a plurality (e.g., panel or bank) of iRNAagents, comparing the information about the target gene to informationabout the plurality of iRNA agents, and selecting one or more of theplurality of iRNA agents for treating the subject. Each of the pluralityof iRNA agents includes a first sequence complementary to a firstvariant target RNA region and a second sequence complementary to asecond variant target RNA region, and the first and second varianttarget RNA regions correspond to first and second variants of the targetgene. The target gene can be an endogenous gene of the subject or aviral gene. The information about the plurality of iRNA agents can bethe sequence of the first or second sequence of one or more of theplurality.

In certain embodiments, at least one of the selected iRNA agentsincludes a sequence capable of hybridizing to an RNA regioncorresponding to the target gene, and at least one of the selected iRNAagents comprises a sequence capable of hybridizing to an RNA regioncorresponding to a variant or mutant of the target gene.

In one aspect, the invention relates to compositions and methods forsilencing genes expressed in the liver, e.g., to treat disorders of orrelated to the liver. An iRNA agent composition of the invention can beone which has been modified to alter distribution in favor of the liver.

In another aspect, the invention relates to iRNA agents that can targetmore than one RNA region, and methods of using and making the iRNAagents. In one embodiment, the RNA is from a gene that is active in theliver, e.g., apoB-100, glucose-6-phosphatase, beta-catenin, or HepatitisC virus (HCV).

In another aspect, an iRNA agent includes a first and second sequencethat are sufficiently complementary to each other to hybridize. Thefirst sequence can be complementary to a first target RNA region and thesecond sequence can be complementary to a second target RNA region. Forexample, the first sequence can be complementary to a first targetapoB-100 RNA region and the second sequence can be complementary to asecond target apoB-100 RNA region.

In one embodiment, the first target RNA region is encoded by a firstgene, e.g., a gene expressed in the liver, and the second target RNAregion is encoded by a second gene, e.g., a second gene expressed in theliver. In another embodiment, the first and second target RNA regionsare different regions of an RNA from a single gene, e.g., a single genethat is at least expressed in the liver. In another embodiment, thefirst and second sequences differ by at least one and no more than sixnucleotides.

In another embodiment, sequence analysis, e.g., to identify commonmutants in the target gene, is used to identify a region of the targetgene that has high variability or mutational frequency. For example,sequence analysis can be used to identify regions of aopB-100 or betacatenin that have high variability or mutational frequency. In anotherembodiment, the region of the target gene having high variability ormutational frequency is identified by obtaining or providing genotypeinformation about the target gene from a population. In particular, thegenotype information can be from a population suffering from a liverdisorder, such as hepatocellular carcinoma or hepatoblastoma.

In another aspect, the invention features a method for reducing apoB-100levels in a subject, e.g., a mammal, such as a human. The methodincludes administering to a subject an iRNA agent which targetsapoB-100. The iRNA agent can be one described here, and can be a dsRNAthat has a sequence that is substantially identical to a sequence of theapoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g.,21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. Inone embodiment, the iRNA is 21 nucleotides in length, and the duplexregion of the iRNA is 19 nucleotides. In another embodiment, the iRNA isgreater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agentwhich targets one of the sequences listed in Tables 5 and 6. In apreferred embodiment it targets both sequences of a palindromic pairprovided in Tables 5 and 6. The most preferred targets are listed indescending order of preferrability, in other words, the more preferredtargets are listed earlier in Tables 5 and 6.

In a preferred embodiment the iRNA agent will include regions, orstrands, which are complementary to a pair in Tables 5 and 6. In apreferred embodiment the iRNA agent will include regions complementaryto the palindromic pairs of Tables 5 and 6 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent willtarget a sequence listed in Tables 5 and 6 but will not be perfectlycomplementary with the target sequence, e.g., it will not becomplementary at at least 1 base pair. Preferably it will have no morethan 1, 2, 3, 4, or 5 bases, in total, or per strand, which do nothybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or5′ overhangs, preferably one or more 3′ overhangs. Overhangs arediscussed in detail elsewhere herein but are preferably about 2nucleotides in length. The overhangs can be complementary to the genesequences being targeted or can be other sequence. TT is a preferredoverhang sequence. The first and second iRNA agent sequences can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

The iRNA agent that targets apoB-100 can be administered in an amountsufficient to reduce expression of apoB-100 mRNA. In one embodiment, theiRNA agent is administered in an amount sufficient to reduce expressionof apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA orprotein. This can be effected, e.g., by selection of an iRNA agent whichspecifically targets the nucleotides subject to RNA editing in theapoB-100 transcript.

The iRNA agent that targets apoB-100 can be administered to a subject,wherein the subject is suffering from a disorder characterized byelevated or otherwise unwanted expression of apoB-100, elevated orotherwise unwanted levels of cholesterol, and/or disregulation of lipidmetabolism. The iRNA agent can be administered to an individual at riskfor the disorder to delay onset of the disorder or a symptom of thedisorder. These disorders include HDL/LDL cholesterol imbalance;dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquiredhyperlipidemia; hypercholestorolemia; statin-resistanthypercholesterolemia; coronary artery disease (CAD) coronary heartdisease (CHD) atherosclerosis. In one embodiment, the iRNA that targetsapoB-100 is administered to a subject suffering from statin-resistanthypercholesterolemia.

The apoB-100 iRNA agent can be administered in an amount sufficient toreduce levels of serum LDL-C and/or HDL-C and/or total cholesterol in asubject. For example, the iRNA is administered in an amount sufficientto decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in thesubject. In one embodiment, the iRNA agent is administered in an amountsufficient to reduce the risk of myocardial infarction the subject.

In a preferred embodiment the iRNA agent is administered repeatedly.Administration of an iRNA agent can be carried out over a range of timeperiods. It can be administered daily, once every few days, weekly, ormonthly. The timing of administration can vary from patient to patient,depending on such factors as the severity of a patient's symptoms. Forexample, an effective dose of an iRNA agent can be administered to apatient once a month for an indefinite period of time, or until thepatient no longer requires therapy. In addition, sustained releasecompositions containing an iRNA agent can be used to maintain arelatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and apoBexpression level are decreased in the liver following administration ofthe apoB iRNA agent. For example, the iRNA agent can be complexed with amoiety that targets the liver, e.g., an antibody or ligand that binds areceptor on the liver.

The iRNA agent, particularly an iRNA agent that targets apoB,beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver,for example by associating, e.g., conjugating the iRNA agent to alipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, orcholesteryl residue (see Table 1). Other lipophilic moieties that can beassociated, e.g., conjugated with the iRNA agent include cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent canbe targeted to the liver by associating, e.g., conjugating, the iRNAagent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. Inanother embodiment, the iRNA agent can be targeted to the liver byassociating, e.g., conjugating, the iRNA agent to a polymeric carriercomplex with sugar residues.

In another embodiment, the iRNA agent can be targeted to the liver byassociating, e.g., conjugating, the iRNA agent to a liposome complexedwith sugar residues. A targeting agent that incorporates a sugar, e.g.,galactose and/or analogues thereof, is particularly useful. These agentstarget, in particular, the parenchymal cells of the liver (see Table 1).In a preferred embodiment, the targeting moiety includes more than onegalactose moiety, preferably two or three. Preferably, the targetingmoiety includes 3 galactose moieties, e.g., spaced about 15 angstromsfrom each other. The targeting moiety can be lactose. A lactose is aglucose coupled to a galactose. Preferably, the targeting moietyincludes three lactoses. The targeting moiety can also beN-Acetyl-Galactosamine, N—Ac-Glucosamine. A mannose, ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

The targeting agent can be linked directly, e.g., covalently or noncovalently, to the iRNA agent, or to another delivery or formulationmodality, e.g., a liposome. E.g., the iRNA agents with or without atargeting moiety can be incorporated into a delivery modality, e.g., aliposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to a lipophilicmolecule to conjugate to an iRNA agent that targets apoB, beta-cateninor glucose-6-phosphatase iRNA targeting agent.

In one embodiment, the iRNA agent has been modified, or is associatedwith a delivery agent, e.g., a delivery agent described herein, e.g., aliposome, which has been modified to alter distribution in favor of theliver. In one embodiment, the modification mediates association with aserum albumin (SA), e.g., a human serum albumin (HSA), or a fragmentthereof.

The iRNA agent, particularly an iRNA agent that targets apoB,beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver,for example by associating, e.g., conjugating the iRNA agent to an SAmolecule, e.g., an HSA molecule, or a fragment thereof. In oneembodiment, the iRNA agent or composition thereof has an affinity for anSA, e.g., HSA, which is sufficiently high such that its levels in theliver are at least 10, 20, 30, 50, or 100% greater in the presence ofSA, e.g., HSA, or is such that addition of exogenous SA will increasedelivery to the liver. These criteria can be measured, e.g., by testingdistribution in a mouse in the presence or absence of exogenous mouse orhuman SA.

The SA, e.g., HSA, targeting agent can be linked directly, e.g.,covalently or noncovalently, to the iRNA agent, or to another deliveryor formulation modality, e.g., a liposome. E.g., the iRNA agents with orwithout a targeting moiety can be incorporated into a delivery modality,e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to an SA, e.g.,an HSA, molecule wherein the iRNA agent is an apoB, beta-catenin orglucose-6-phosphatase iRNA targeting agent.

In another aspect, the invention features, a method for reducingglucose-6-phosphatase levels in a subject, e.g., a mammal, such as ahuman. The method includes administering to a subject an iRNA agentwhich targets glucose-6-phosphatase. The iRNA agent can be a dsRNA thathas a sequence that is substantially identical to a sequence of theglucose-6-phosphatase gene.

In a preferred embodiment, the subject is treated with an iRNA agentwhich targets one of the sequences listed in Table 7. In a preferredembodiment it targets both sequences of a palindromic pair provided inTable 7. The most preferred targets are listed in descending order ofpreferrability, in other words, the more preferred targets are listedearlier in Table 7.

In a preferred embodiment the iRNA agent will include regions, orstrands, which are complementary to a pair in Table 7. In a preferredembodiment the iRNA agent will include regions complementary to thepalindromic pairs of Table 7 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent willtarget a sequence listed in Table 7 but will not be perfectlycomplementary with the target sequence, e.g., it will not becomplementary at at least 1 base pair. Preferably it will have no morethan 1, 2, 3, 4, or 5 bases, in total, or per strand, which do nothybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or5′ overhangs, preferably one or more 3′ overhangs. Overhangs arediscussed in detail elsewhere herein but are preferably about 2nucleotides in length. The overhangs can be complementary to the genesequences being targeted or can be other sequence. TT is a preferredoverhang sequence. The first and second iRNA agent sequences can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

Table 7 refers to sequences from human glucose-6-phosphatase. Table 8refers to sequences from rat glucose-6-phosphatase. The sequences fromtable 8 can be used, e.g., in experiments with rats or cultured ratcells.

In a preferred embodiment iRNA agent can have any architecture, e.g.,architecture described herein. E.g., it can be incorporated into an iRNAagent having an overhang structure, overall length, hairpin vs.two-strand structure, as described herein. In addition, monomers otherthan naturally occurring ribonucleotides can be used in the selectediRNA agent.

The iRNA that targets glucose-6-phosphatase can be administered in anamount sufficient to reduce expression of glucose-6-phosphatase mRNA.

The iRNA that targets glucose-6-phosphatase can be administered to asubject to inhibit hepatic glucose production, for the treatment ofglucose-metabolism-related disorders, such as diabetes, e.g.,type-2-diabetes mellitus. The iRNA agent can be administered to anindividual at risk for the disorder to delay onset of the disorder or asymptom of the disorder.

In other embodiments, iRNA agents having sequence similarity to thefollowing genes can also be used to inhibit hepatic glucose production.These other genes include “forkhead homologue in rhabdomyosarcoma(FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-GammaCoactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatelocator; glucokinase inhibitory regulatory protein; andphosphoenolpyruvate carboxykinase (PEPCK).

In one embodiment, the iRNA agent can be targeted to the liver, and RNAexpression levels of the targeted genes are decreased in the liverfollowing administration of the iRNA agent.

The iRNA agent can be one described herein, and can be a dsRNA that hasa sequence that is substantially identical to a sequence of a targetgene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23nucleotides. Preferably, the iRNA is 21 nucleotides in length. In oneembodiment, the iRNA is 21 nucleotides in length, and the duplex regionof the iRNA is 19 nucleotides. In another embodiment, the iRNA isgreater than 30 nucleotides in length

In another aspect, the invention features a method for reducingbeta-catenin levels in a subject, e.g., a mammal, such as a human. Themethod includes administering to a subject an iRNA agent that targetsbeta-catenin. The iRNA agent can be one described herein, and can be adsRNA that has a sequence that is substantially identical to a sequenceof the beta-catenin gene. The iRNA can be less than 30 nucleotides inlength, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotidesin length. In one embodiment, the iRNA is 21 nucleotides in length, andthe duplex region of the iRNA is 19 nucleotides. In another embodiment,the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agentwhich targets one of the sequences listed in Table 9. In a preferredembodiment it targets both sequences of a palindromic pair provided inTable 9. The most preferred targets are listed in descending order ofpreferrability, in other words, the more preferred targets are listedearlier in Table 9.

In a preferred embodiment, the subject is treated with an iRNA agentwhich targets one of the sequences listed in Table 9. In a preferredembodiment it targets both sequences of a palindromic pair provided inTable 9. The most preferred targets are listed in descending order ofpreferrability, in other words, the more preferred targets are listedearlier in Table 9.

In a preferred embodiment the iRNA agent will include regions, orstrands, which are complementary to a pair in Table 9. In a preferredembodiment the iRNA agent will include regions complementary to thepalindromic pairs of Table 9 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent willtarget a sequence listed in Table 9 but will not be perfectlycomplementary with the target sequence, e.g., it will not becomplementary at at least 1 base pair. Preferably it will have no morethan 1, 2, 3, 4, or 5 bases, in total, or per strand, which do nothybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or5′ overhangs, preferably one or more 3′ overhangs. Overhangs arediscussed in detail elsewhere herein but are preferably about 2nucleotides in length. The overhangs can be complementary to the genesequences being targeted or can be other sequence. TT is a preferredoverhang sequence. The first and second iRNA agent sequences can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

The iRNA agent that targets beta-catenin can be administered in anamount sufficient to reduce expression of beta-catenin mRNA. In oneembodiment, the iRNA agent is administered in an amount sufficient toreduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%,10%, 15%, 20%).

The iRNA agent that targets beta-catenin can be administered to asubject, wherein the subject is suffering from a disorder characterizedby unwanted cellular proliferation in the liver or of liver tissue,e.g., metastatic tissue originating from the liver. Examples include, abenign or malignant disorder, e.g., a cancer, e.g., a hepatocellularcarcinoma (HCC), hepatic metastasis, or hepatoblastoma.

The iRNA agent can be administered to an individual at risk for thedisorder to delay onset of the disorder or a symptom of the disorder

In a preferred embodiment the iRNA agent is administered repeatedly.Administration of an iRNA agent can be carried out over a range of timeperiods. It can be administered daily, once every few days, weekly, ormonthly. The timing of administration can vary from patient to patient,depending on such factors as the severity of a patient's symptoms. Forexample, an effective dose of an iRNA agent can be administered to apatient once a month for an indefinite period of time, or until thepatient no longer requires therapy. In addition, sustained releasecompositions containing an iRNA agent can be used to maintain arelatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, andbeta-catenin expression level are decreased in the liver followingadministration of the beta-catenin iRNA agent. For example, the iRNAagent can be complexed with a moiety that targets the liver, e.g., anantibody or ligand that binds a receptor on the liver.

In another aspect, the invention provides methods to treat liverdisorders, e.g., disorders characterized by unwanted cell proliferation,hematological disorders, disorders characterized by inflammationdisorders, and metabolic or viral diseases or disorders of the liver. Aproliferation disorder of the liver can be, for example, a benign ormalignant disorder, e.g., a cancer, e.g, a hepatocellular carcinoma(HCC), hepatic metastasis, or hepatoblastoma. A hepatic hematology orinflammation disorder can be a disorder involving clotting factors, acomplement-mediated inflammation or a fibrosis, for example. Metabolicdiseases of the liver can include dyslipidemias, and irregularities inglucose regulation. Viral diseases of the liver can include hepatitis Cor hepatitis B. In one embodiment, a liver disorder is treated byadministering one or more iRNA agents that have a sequence that issubstantially identical to a sequence in a gene involved in the liverdisorder.

In one embodiment an iRNA agent to treat a liver disorder has a sequencewhich is substantially identical to a sequence of the beta-catenin orc-jun gene. In another embodiment, such as for the treatment ofhepatitis C or hepatitis B, the iRNA agent can have a sequence that issubstantially identical to a sequence of a gene of the hepatitis C virusor the hepatitis B virus, respectively. For example, the iRNA agent cantarget the 5′ core region of HCV. This region lies just downstream ofthe ribosomal toe-print straddling the initiator methionine.Alternatively, an iRNA agent of the invention can target any one of thenonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatmentof hepatitis B, an iRNA agent can target the protein X (HBx) gene, forexample.

In a preferred embodiment, the subject is treated with an iRNA agentwhich targets one of the sequences listed in Table 10. In a preferredembodiment it targets both sequences of a palindromic pair provided inTable 10. The most preferred targets are listed in descending order ofpreferrability, in other words, the more preferred targets are listedearlier in Table 10.

In a preferred embodiment the iRNA agent will include regions, orstrands, which are complementary to a pair in Table 10. In a preferredembodiment the iRNA agent will include regions complementary to thepalindromic pairs of Table 10 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent willtarget a sequence listed in Table 10, but will not be perfectlycomplementary with the target sequence, e.g., it will not becomplementary at at least 1 base pair. Preferably it will have no morethan 1, 2, 3, 4, or 5 bases, in total, or per strand, which do nothybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or5′ overhangs, preferably one or more 3′ overhangs. Overhangs arediscussed in detail elsewhere herein but are preferably about 2nucleotides in length. The overhangs can be complementary to the genesequences being targeted or can be other sequence. TT is a preferredoverhang sequence. The first and second iRNA agent sequences can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In another aspect, an iRNA agent can be administered to modulate bloodclotting, e.g., to reduce the tendency to form a blood clot. In apreferred embodiment the iRNA agent targets Factor V expression,preferably in the liver. One or more iRNA agents can be used to target awild type allele, a mutant allele, e.g., the Leiden Factor V allele, orboth. Such administration can be used to treat or prevent venousthrombosis, e.g., deep vein thrombosis or pulmonary embolism, or anotherdisorder caused by elevated or otherwise unwanted expression of FactorV, in, e.g., the liver. In one embodiment the iRNA agent can treat asubject, e.g., a human who has Factor V Leiden or other genetic traitassociated with an unwanted tendency to form blood clots.

In a preferred embodiment administration of an iRNA agent which targetsFactor V is with the administration of a second treatment, e.g, atreatment which reduces the tendency of the blood to clot, e.g., theadministration of heparin or of a low molecular weight heparin.

In one embodiment, the iRNA agent that targets Factor V can be used as aprophylaxis in patients, e.g., patients with Factor V Leiden, who areplaced at risk for a thrombosis, e.g., those about to undergo surgery,in particular those about to undergo high-risk surgical procedures knownto be associated with formation of venous thrombosis, those about toundergo a prolonged period of relative inactivity, e.g., on a motorvehicle, train or airplane flight, e.g., a flight or other trip lastingmore than three or five hours. Such a treatment can be an adjunct to thetherapeutic use of low molecular weight (LMW) heparin prophylaxis.

In another embodiment, the iRNA agent that targets Factor V can beadministered to patients with Factor V Leiden to treat deep veinthrombosis (DVT) or pulmonary embolism (PE). Such a treatment can be anadjunct to (or can replace) therapeutic uses of heparin or coumadin. Thetreatment can be administered by inhalation or generally by pulmonaryroutes.

In a preferred embodiment, an iRNA agent administered to treat a liverdisorder is targeted to the liver. For example, the iRNA agent can becomplexed with a targeting moiety, e.g., an antibody or ligand thatrecognizes a liver-specific receptor.

The invention also includes preparations, including substantially pureor pharmaceutically acceptable preparations of iRNA agents which silenceany of the genes discussed herein and in particular for any of apoB-100,glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genesdiscussed herein.

The methods and compositions of the invention, e.g., the methods andcompositions to treat diseases and disorders of the liver describedherein, can be used with any of the iRNA agents described. In addition,the methods and compositions of the invention can be used for thetreatment of any disease or disorder described herein, and for thetreatment of any subject, e.g., any animal, any mammal, such as anyhuman.

In another aspect, the invention features, a method of selecting twosequences or strands for use in an iRNA agent. The method includes:

providing a first candidate sequence and a second candidate sequence;

determining the value of a parameter which is a function of the numberof palindromic pairs between the first and second sequence, wherein apalindromic pair is a nucleotide on said first sequence which, when thesequences are aligned in anti-parallel orientation, will hybridize witha nucleotide on said second sequence;

comparing the number with a predetermined reference value, and if thenumber has a predetermined relationship with the reference, e.g., if itis the same or greater, selecting the sequences for use in an iRNAagent. In most cases each of the two sequences will be completelycomplementary with a target sequence (though as described elsewhereherein that may not always be the case, there may not be perfectcomplementarity with one or both of the target sequences) and will havesufficient complementarity with each other to form a duplex. Theparameter can be derived e.g., by directly determining the number ofpalindromic pairs, e.g., by inspection or by the use of a computerprogram which compares or analyses sequence. The parameter can also bedetermined less directly, and include e.g., calculation of ormeasurement of the Tm or other value related to the free energy ofassociation or dissociation of a duplex.

In a preferred embodiment the determination can be performed on a targetsequence, e.g., a genomic sequence. In such embodiments the selectedsequence is converted to its complement in the iRNA agent.

In a preferred embodiment the first and second sequences are selectedfrom the sequence of a single target gene. In other embodiments thefirst sequence is selected from the sequence of a first target gene andthe second sequence is selected from the target of a second target gene.

In a preferred embodiment the method includes comparing blocks ofsequence, e.g., blocks which are between 15 and 25 nucleotides inlength, and preferably 19, 20, or 21, and most preferably 19 nucleotidesin length, to determine if they are suitable for use, e.g., if theypossess sufficient palindromic pairs.

In a preferred embodiment the first and second sequences are dividedinto a plurality of regions, e.g., terminal regions and a middle regiondisposed between the terminal regions and where in the reference value,or the predetermined relationship to the reference value, is differentfor at least two regions. E.g., the first and second sequences, whenaligned in anti-parallel orientation, are divided into terminal regionseach of a selected number of base pairs, e.g., 2, 3, 4, 5, or 6, and amiddle region, and the reference value for the terminal regions ishigher than for the middle regions. In other words, a higher number orproportion of palindromic pairs is required in the terminal regions.

In a preferred embodiment the first and second sequences are genesequences thus the complements of the sequences will be used in a iRNAagent.

In a preferred embodiment hybridize means a classical Watson-Crickpairing. In other embodiments hybridize can include non-Watson-Crickparing, e.g., parings seen in micro RNA precursors.

In a preferred embodiment the method includes the addition ofnucleotides to form overhangs, e.g., 3′ or 5′ overhangs, preferably oneor more 3′ overhangs. Overhangs are discussed in detail elsewhere hereinbut are preferably about 2 nucleotides in length. The overhangs can becomplementary to the gene sequences being targeted or can be othersequence. TT is a preferred overhang sequence. The first and second iRNAagent sequences can also be joined, e.g., by additional bases to form ahairpin, or by other non-base linkers.

In a preferred embodiment the method is used to select all or part of aiRNA agent. The selected sequences can be incorporated into an iRNAagent having any architecture, e.g., an architecture described herein.E.g., it can be incorporated into an iRNA agent having an overhangstructure, overall length, hairpin vs. two-strand structure, asdescribed herein. In addition, monomers other than naturally occurringribonucleotides can be used in the selected iRNA agent.

Preferred iRNA agents of this method will target genes expressed in theliver, e.g., one of the genes disclosed herein, e.g., apo B, Betacatenin, an HVC gene, or glucose 6 phosphatase.

In another aspect, the invention features, an iRNA agent, determined,made, or selected by a method described herein.

The methods and compositions of the invention, e.g., the methods andiRNA compositions to treat liver-based diseases described herein, can beused with any dosage and/or formulation described herein, as well aswith any route of administration described herein.

The invention also provides for the use of an iRNA agent which includesmonomers which can form other than a canonical Watson-Crick pairing withanother monomer, e.g., a monomer on another strand.

The use of “other than canonical Watson-Crick pairing” between monomersof a duplex can be used to control, often to promote, melting of all orpart of a duplex. The iRNA agent can include a monomer at a selected orconstrained position that results in a first level of stability in theiRNA agent duplex (e.g., between the two separate molecules of a doublestranded iRNA agent) and a second level of stability in a duplex betweena sequence of an iRNA agent and another sequence molecule, e.g., atarget or off-target sequence in a subject. In some cases the secondduplex has a relatively greater level of stability, e.g., in a duplexbetween an anti-sense sequence of an iRNA agent and a target mRNA. Inthis case one or more of the monomers, the position of the monomers inthe iRNA agent, and the target sequence (sometimes referred to herein asthe selection or constraint parameters), are selected such that the iRNAagent duplex is has a comparatively lower free energy of association(which while not wishing to be bound by mechanism or theory, is believedto contribute to efficacy by promoting disassociation of the duplex iRNAagent in the context of the RISC) while the duplex formed between ananti-sense targeting sequence and its target sequence, has a relativelyhigher free energy of association (which while not wishing to be boundby mechanism or theory, is believed to contribute to efficacy bypromoting association of the anti-sense sequence and the target RNA).

In other cases the second duplex has a relatively lower level ofstability, e.g., in a duplex between a sense sequence of an iRNA agentand an off-target mRNA. In this case one or more of the monomers, theposition of the monomers in the iRNA agent, and an off-target sequence,are selected such that the iRNA agent duplex is has a comparativelyhigher free energy of association while the duplex formed between asense targeting sequence and its off-target sequence, has a relativelylower free energy of association (which while not wishing to be bound bymechanism or theory, is believed to reduce the level of off-targetsilencing by contribute to efficacy by promoting disassociation of theduplex formed by the sense strand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property ofhaving a first stability for the intra-iRNA agent duplex and a secondstability for a duplex formed between a sequence from the iRNA agent andanother RNA, e.g., a target mRNA. As discussed above, this can beaccomplished by judicious selection of one or more of the monomers at aselected or constrained position, the selection of the position in theduplex to place the selected or constrained position, and selection ofthe sequence of a target sequence (e.g., the particular region of atarget gene which is to be targeted). The iRNA agent sequences whichsatisfy these requirements are sometimes referred herein as constrainedsequences. Exercise of the constraint or selection parameters can be,e.g., by inspection, or by computer assisted methods. Exercise of theparameters can result in selection of a target sequence and ofparticular monomers to give a desired result in terms of the stability,or relative stability, of a duplex.

Thus, in one aspect, the invention features, an iRNA agent whichincludes: a first sequence which targets a first target region and asecond sequence which targets a second target region. The first andsecond sequences have sufficient complementarity to each other tohybridize, e.g., under physiological conditions, e.g., underphysiological conditions but not in contact with a helicase or otherunwinding enzyme. In a duplex region of the iRNA agent, at a selected orconstrained position, the first target region has a first monomer, andthe second target region has a second monomer. The first and secondmonomers occupy complementary or corresponding positions. One, andpreferably both monomers are selected such that the stability of thepairing of the monomers contribute to a duplex between the first andsecond sequence will differ form the stability of the pairing betweenthe first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequencemay be required as well) such that they form a pairing in the iRNA agentduplex which has a lower free energy of dissociation, and a lower Tm,than will be possessed by the paring of the monomer with itscomplementary monomer in a duplex between the iRNA agent sequence and atarget RNA duplex.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes,including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g.,21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length andthere is a duplex region of about 19 pairs. In one embodiment, the iRNAis 21 nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

The monomers can be selected such that: first and second monomers arenaturally occurring ribonucleotides, or modified ribonucleotides havingnaturally occurring bases, and when occupying complementary sites eitherdo not pair and have no substantial level of H-bonding, or form a noncanonical Watson-Crick pairing and form a non-canonical pattern of Hbonding, which usually have a lower free energy of dissociation thanseen in a canonical Watson-Crick pairing, or otherwise pair to give afree energy of association which is less than that of a preselectedvalue or is less, e.g., than that of a canonical pairing. When one (orboth) of the iRNA agent sequences duplexes with a target, the first (orsecond) monomer forms a canonical Watson-Crick pairing with the base inthe complementary position on the target, or forms a non canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the paring in the iRNA agent. The classicalWatson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonicalWatson-Crick pairings are known in the art and can include, U-U, G-G,G-Atrans, G-Acis, and GU.

The monomer in one or both of the sequences is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theother sequence which minimizes stability (e.g., the H bonding formedbetween the monomer at the selected site in the one sequence and itsmonomer at the corresponding site in the other sequence are less stablethan the H bonds formed by the monomer one (or both) of the sequenceswith the respective target sequence. The monomer in one or both strandsis also chosen to promote stability in one or both of the duplexes madeby a strand and its target sequence. E.g., one or more of the monomersand the target sequences are selected such that at the selected orconstrained position, there is are no H bonds formed, or a non canonicalpairing is formed in the iRNA agent duplex, or otherwise they otherwisepair to give a free energy of association which is less than that of apreselected value or is less, e.g., than that of a canonical pairing,but when one (or both) sequences form a duplex with the respectivetarget, the pairing at the selected or constrained site is a canonicalWatson-Crick pairing.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the a sequence and the intended target will not be destabilized.

By way of example:

the monomer at the selected site in the first sequence includes an A (ora modified base which pairs with T), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G. These will beuseful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

the monomer at the selected site in the first sequence includes U (or amodified base which pairs with A), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., U or G. Thesewill be useful in applications wherein the target sequence for the firstsequence has a T at the selected position. In embodiments where bothtarget duplexes are stabilized it is useful wherein the target sequencefor the second strand has a monomer which will form a canonicalWatson-Crick pairing with the monomer selected for the selected positionin the second strand.

The monomer at the selected site in the first sequence includes a G (ora modified base which pairs with C), and the monomer in at the selectedposition in the second sequence is chosen from a monomer which will notpair or which will form a non-canonical pairing, e.g., G, Acis, Atrans,or U. These will be useful in applications wherein the target sequencefor the first sequence has a T at the selected position. In embodimentswhere both target duplexes are stabilized it is useful wherein thetarget sequence for the second strand has a monomer which will form acanonical Watson-Crick pairing with the monomer selected for theselected position in the second strand.

The monomer at the selected site in the first sequence includes a C (ora modified base which pairs with G), and the monomer in at the selectedposition in the second sequence is chosen a monomer which will not pairor which will form a non-canonical pairing. These will be useful inapplications wherein the target sequence for the first sequence has a Tat the selected position. In embodiments where both target duplexes arestabilized it is useful wherein the target sequence for the secondstrand has a monomer which will form a canonical Watson-Crick pairingwith the monomer selected for the selected position in the secondstrand.

In another embodiment a non-naturally occurring or modified monomer ormonomers are chosen such that when a non-naturally occurring or modifiedmonomer occupies a positions at the selected or constrained position inan iRNA agent they exhibit a first free energy of dissociation and whenone (or both) of them pairs with a naturally occurring monomer, the pairexhibits a second free energy of dissociation, which is usually higherthan that of the pairing of the first and second monomers. E.g., whenthe first and second monomers occupy complementary positions they eitherdo not pair and have no substantial level of H-bonding, or form a weakerbond than one of them would form with a naturally occurring monomer, andreduce the stability of that duplex, but when the duplex dissociates atleast one of the strands will form a duplex with a target in which theselected monomer will promote stability, e.g., the monomer will form amore stable pair with a naturally occurring monomer in the targetsequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomerswill pair very poorly and will minimize stability. However, a duplex isformed between 2 amino A and the U of a naturally occurring target, or aduplex is between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable. Thisis shown in the FIG. 1.

The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) isexemplary. In another embodiment, the monomer at the selected positionin the sense strand can be a universal pairing moiety. A universalpairing agent will form some level of H bonding with more than one andpreferably all other naturally occurring monomers. An example of auniversal pairing moiety is a monomer which includes 3-nitro pyrrole.(Examples of other candidate universal base analogs can be found in theart, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated byreference. Examples can also be found in the section on Universal Basesbelow.) In these cases the monomer at the corresponding position of theanti-sense strand can be chosen for its ability to form a duplex withthe target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes:a sense sequence, which preferably does not target a sequence in asubject, and an anti-sense sequence, which targets a target gene in asubject. The sense and anti-sense sequences have sufficientcomplementarity to each other to hybridize hybridize, e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme. In a duplex regionof the iRNA agent, at a selected or constrained position, the monomersare selected such that:

the monomer in the sense sequence is selected such that, it does notpair, or forms a pair with its corresponding monomer in the anti-sensestrand which minimizes stability (e.g., the H bonding formed between themonomer at the selected site in the sense strand and its monomer at thecorresponding site in the anti-sense strand are less stable than the Hbonds formed by the monomer of the anti-sense sequence and its canonicalWatson-Crick partner or, if the monomer in the anti-sense strandincludes a modified base, the natural analog of the modified base andits canonical Watson-Crick partner);

the monomer is in the corresponding position in the anti-sense strand isselected such that it maximizes the stability of a duplex it forms withthe target sequence, e.g., it forms a canonical Watson-Crick paring withthe monomer in the corresponding position on the target stand;

optionally, the monomer in the sense sequence is selected such that, itdoes not pair, or forms a pair with its corresponding monomer in theanti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the followingeffects: it will destabilize the iRNA agent duplex, it will destabilizeinteractions between the sense sequence and unintended target sequences,sometimes referred to as off-target sequences, and duplex interactionsbetween the anti-sense strand and the intended target will not bedestabilized.

The constraint placed upon the monomers can be applied at a selectedsite or at more than one selected site. By way of example, theconstraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positionsin the iRNA agent duplex. E.g., a constrained or selected site can bepresent within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of aduplexed sequence. A constrained or selected site can be present in themiddle of the duplex region, e.g., it can be more than 3, 4, 5, or 6,positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes,including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture(architecture refers to one or more of overall length, length of aduplex region, the presence, number, location, or length of overhangs,sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g.,21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length andthere is a duplex region of about 19 pairs. In one embodiment, the iRNAis 21 nucleotides in length, and the duplex region of the iRNA is 19nucleotides. In another embodiment, the iRNA is greater than 30nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have,mismatches, in addition to the selected or constrained site or sites.Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which donot form canonical Watson-Crick pairs or which do not hybridize.Overhangs are discussed in detail elsewhere herein but are preferablyabout 2 nucleotides in length. The overhangs can be complementary to thegene sequences being targeted or can be other sequence. TT is apreferred overhang sequence. The first and second iRNA agent sequencescan also be joined, e.g., by additional bases to form a hairpin, or byother non-base linkers.

One or more selection or constraint parameters can be exercised suchthat: monomers at the selected site in the sense and anti-sensesequences are both naturally occurring ribonucleotides, or modifiedribonucleotides having naturally occurring bases, and when occupyingcomplementary sites in the iRNA agent duplex either do not pair and haveno substantial level of H-bonding, or form a non-canonical Watson-Crickpairing and thus form a non-canonical pattern of H bonding, whichgenerally have a lower free energy of dissociation than seen in aWatson-Crick pairing, or otherwise pair to give a free energy ofassociation which is less than that of a preselected value or is less,e.g., than that of a canonical pairing. When one, usually the anti-sensesequence of the iRNA agent sequences forms a duplex with anothersequence, generally a sequence in the subject, and generally a targetsequence, the monomer forms a classic Watson-Crick pairing with the basein the complementary position on the target, or forms a non-canonicalWatson-Crick pairing having a higher free energy of dissociation and ahigher Tm than seen in the paring in the iRNA agent. Optionally, whenthe other sequence of the iRNA agent, usually the sense sequences formsa duplex with another sequence, generally a sequence in the subject, andgenerally an off-target sequence, the monomer fails to forms a canonicalWatson-Crick pairing with the base in the complementary position on theoff target sequence, e.g., it forms or forms a non-canonicalWatson-Crick pairing having a lower free energy of dissociation and alower Tm.

By way of example:

the monomer at the selected site in the anti-sense stand includes an A(or a modified base which pairs with T), the corresponding monomer inthe target is a T, and the sense strand is chosen from a base which willnot pair or which will form a noncanonical pair, e.g., G;

the monomer at the selected site in the anti-sense stand includes a U(or a modified base which pairs with A), the corresponding monomer inthe target is an A, and the sense strand is chosen from a monomer whichwill not pair or which will form a non-canonical pairing, e.g., U or G;

the monomer at the selected site in the anti-sense stand includes a C(or a modified base which pairs with G), the corresponding monomer inthe target is a G, and the sense strand is chosen a monomer which willnot pair or which will form a non-canonical pairing, e.g., G, A_(cis),A_(trans), or U; or

the monomer at the selected site in the anti-sense stand includes a G(or a modified base which pairs with C), the corresponding monomer inthe target is a C, and the sense strand is chosen from a monomer whichwill not pair or which will form a non-canonical pairing.

In another embodiment a non-naturally occurring or modified monomer ormonomers is chosen such that when it occupies complementary a positionin an iRNA agent they exhibit a first free energy of dissociation andwhen one (or both) of them pairs with a naturally occurring monomer, thepair exhibits a second free energy of dissociation, which is usuallyhigher than that of the pairing of the first and second monomers. E.g.,when the first and second monomers occupy complementary positions theyeither do not pair and have no substantial level of H-bonding, or form aweaker bond than one of them would form with a naturally occurringmonomer, and reduce the stability of that duplex, but when the duplexdissociates at least one of the strands will form a duplex with a targetin which the selected monomer will promote stability, e.g., the monomerwill form a more stable pair with a naturally occurring monomer in thetarget sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thiopyrimidine analog of U or T. As is discussed above, when placed incomplementary positions of the iRNA agent these monomers will pair verypoorly and will minimize stability. However, a duplex is formed between2 amino A and the U of a naturally occurring target, or a duplex isformed between 2-thio U and the A of a naturally occurring target or2-thio T and the A of a naturally occurring target will have arelatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be auniversal pairing moiety. A universal pairing agent will form some levelof H bonding with more than one and preferably all other naturallyoccurring monomers. An examples of a universal pairing moiety is amonomer which includes 3-nitro pyrrole. Examples of other candidateuniversal base analogs can be found in the art, e.g., in Loakes, 2001,NAR 29: 2437-2447, hereby incorporated by reference. In these cases themonomer at the corresponding position of the anti-sense strand can bechosen for its ability to form a duplex with the target and can include,e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes:

a sense sequence, which preferably does not target a sequence in asubject, and an anti-sense sequence, which targets a plurality of targetsequences in a subject, wherein the targets differ in sequence at only 1or a small number, e.g., no more than 5, 4, 3 or 2 positions. The senseand anti-sense sequences have sufficient complementarity to each otherto hybridize, e.g., under physiological conditions, e.g., underphysiological conditions but not in contact with a helicase or otherunwinding enzyme. In the sequence of the anti-sense strand of the iRNAagent is selected such that at one, some, or all of the positions whichcorrespond to positions that differ in sequence between the targetsequences, the anti-sense strand will include a monomer which will formH-bonds with at least two different target sequences. In a preferredexample the anti-sense sequence will include a universal or promiscuousmonomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A,2-thio U or 2-thio T, or other universal base referred to herein.

In a preferred embodiment the iRNA agent targets repeated sequences(which differ at only one or a small number of positions from eachother) in a single gene, a plurality of genes, or a viral genome, e.g.,the HCV genome.

An embodiment is illustrated in the FIGS. 2 and 3.

In another aspect, the invention features, determining, e.g., bymeasurement or calculation, the stability of a pairing between monomersat a selected or constrained position in the iRNA agent duplex, andpreferably determining the stability for the corresponding pairing in aduplex between a sequence form the iRNA agent and another RNA, e.g., atarget sequence. The determinations can be compared. An iRNA agent thusanalyzed can be used in the development of a further modified iRNA agentor can be administered to a subject. This analysis can be performedsuccessively to refine or design optimized iRNA agents.

In another aspect, the invention features, a kit which includes one ormore of the following an iRNA described herein, a sterile container inwhich the iRNA agent is disclosed, and instructions for use.

In another aspect, the invention features, an iRNA agent containing aconstrained sequence made by a method described herein. The iRNA agentcan target one or more of the genes referred to herein.

iRNA agents having constrained or selected sites, e.g., as describedherein, can be used in any way described herein. Accordingly, they iRNAagents having constrained or selected sites, e.g., as described herein,can be used to silence a target, e.g., in any of the methods describedherein and to target any of the genes described herein or to treat anyof the disorders described herein. iRNA agents having constrained orselected sites, e.g., as described herein, can be incorporated into anyof the formulations or preparations, e.g., pharmaceutical or sterilepreparations described herein. iRNA agents having constrained orselected sites, e.g., as described herein, can be administered by any ofthe routes of administration described herein.

The term “other than canonical Watson-Crick pairing” as used herein,refers to a pairing between a first monomer in a first sequence and asecond monomer at the corresponding position in a second sequence of aduplex in which one or more of the following is true: (1) there isessentially no pairing between the two, e.g., there is no significantlevel of H bonding between the monomers or binding between the monomersdoes not contribute in any significant way to the stability of theduplex; (2) the monomers are a non-canonical paring of monomers having anaturally occurring bases, i.e., they are other than A-T, A-U, or G-C,and they form monomer-monomer H bonds, although generally the H bondingpattern formed is less strong than the bonds formed by a canonicalpairing; or (3) at least one of the monomers includes a non-naturallyoccurring bases and the H bonds formed between the monomers is,preferably formed is less strong than the bonds formed by a canonicalpairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to a sequence other thanthe sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair betweendifluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu,D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-endproofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.;Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray,T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specificallyand efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.;Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isosterefor deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org.Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination.Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.;Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

-   (1. Efforts toward the expansion of the genetic alphabet:    Information storage and replication with unnatural hydrophobic base    pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2.    Rational design of an unnatural base pair with increased kinetic    selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.;    Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNAwith three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization ofinterbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.;McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000,122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNApolymerase recognition of a highly stable, self-pairing hydrophobicbase. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.;Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shapecomplementary base couple: Interstrand stacking as the stabilitydetermining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew.Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNAwith intra-duplex metal complexes. Weizman, H; Tor, Y. J. Am. Chem.Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes.Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity ofneighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W.Organic Lett., 2001, 3, 1977-1980. 2. Effect of the1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability ofDNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R.Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure anddeoxyribonucleic acid sequencing with a universal nucleosides:1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang,P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995,117, 1201-1209)

(Model studies directed toward a general triplex DNA recognition scheme:a novel DNA base that binds a CG base-pair in an organic solvent.Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside.J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine.Nakatani, K; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122,2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into singleguanine bulge as evidenced by photooxidation of GC doublet, Nakatani,K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001,11, 335-337)

Other universal bases can have the following formulas:

wherein:

Q is N or CR⁴⁴;

Q′ is N or CR⁴⁵;

Q″ is N or CR⁴⁷;

Q′″ is N or CR⁴⁹;

Q^(iv) is N or CR⁵⁰;

R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁵ forms —OCH₂O—;

R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms —OCH₂O—;

R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms —OCH₂O—;

R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms —OCH₂O—;

R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈heterocyclyl, or when taken together with R⁴⁷ forms —OCH₂O—;

R⁴⁹, R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴,R⁶⁵, R⁶⁶, R⁶⁷, R⁶⁸, R⁶⁹, R⁷⁰, R⁷¹, and R⁷² are each independentlyselected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂,NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o);

R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when takentogether with R⁵⁶ forms a fused aromatic ring which may be optionallysubstituted;

R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b),or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when takentogether with R⁵⁵ forms a fused aromatic ring which may be optionallysubstituted;

R¹⁷ is halo, NH₂, NHR^(b), or NR^(b)R^(c);

R^(b) is C₁-C₆ alkyl or a nitrogen protecting group;

R^(c) is C₁-C₆ alkyl; and

R^(o) is alkyl optionally substituted with halo, hydroxy, nitro,protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₂-C₆alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷,or NC(O)R^(o).

Examples of universal bases include:

In one aspect, the invention features methods of producing iRNA agents,e.g., sRNA agents, e.g. an sRNA agent described herein, having theability to mediate RNAi. These iRNA agents can be formulated foradministration to a subject.

In another aspect, the invention features a method of administering aniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to asubject (e.g., a human subject). The

method includes administering a unit dose of the iRNA agent, e.g., asRNA agent, e.g., double stranded sRNA agent that (a) thedouble-stranded part is 19-25 nucleotides (nt) long, preferably 21-23nt, (b) is complementary to a target RNA (e.g., an endogenous orpathogen target RNA), and, optionally, (c) includes at least one 3′overhang 1-5 nucleotide long. In one embodiment, the unit dose is lessthan 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1,0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kgof bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15,7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous or intramuscular), an inhaled dose, or a topicalapplication. Particularly preferred dosages are less than 2, 1, or 0.1mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequentlythan once a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than aniRNA agent, e.g., other than a double-stranded iRNA agent, or sRNAagent. In another embodiment, the subject has atherosclerosis and theeffective dose of an iRNA agent, e.g., a double-stranded iRNA agent, orsRNA agent, is administered in combination with, e.g., after surgicalintervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof). The maintenance dose or doses are generally lower than theinitial dose, e.g., one-half less of the initial dose. A maintenanceregimen can include treating the subject with a dose or doses rangingfrom 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1,0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenancedoses are preferably administered no more than once every 5, 10, or 30days.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

The inventors have discovered that iRNA agents described herein can beadministered to mammals, particularly large mammals such as nonhumanprimates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, composition is parenteral,e.g. intravenous (e.g., as a bolus or as a diffusible infusion),intradermal, intraperitoneal, intramuscular, intrathecal,intraventricular, intracranial, subcutaneous, transmucosal, buccal,sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary,intranasal, urethral or ocular. Administration can be provided by thesubject or by another person, e.g., a health care provider. Themedication can be provided in measured doses or in a dispenser thatdelivers a metered dose. Selected modes of delivery are discussed inmore detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes a an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAagent described herein, e.g., a iRNA agent having a double strandedregion of less than 40, and preferably less than 30 nucleotides andhaving one or two 1-3 nucleotide single strand 3′ overhangs can beadministered rectally, e.g., introduced through the rectum into thelower or upper colon. This approach is particularly useful in thetreatment of, inflammatory disorders, disorders characterized byunwanted cell proliferation, e.g., polyps, or colon cancer.

In some embodiments the medication is delivered to a site in the colonby introducing a dispensing device, e.g., a flexible, camera-guideddevice similar to that used for inspection of the colon or removal ofpolyps, which includes means for delivery of the medication.

In one embodiment, the rectal administration of the iRNA agent is bymeans of an enema. The iRNA agent of the enema can be dissolved in asaline or buffered solution.

In another embodiment, the rectal administration is by means of asuppository. The suppository can include other ingredients, e.g., anexcipient, e.g., cocoa butter or hydropropylmethylcellulose.

The invention also provides methods, compositions, and kits for oraldelivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAdescribed herein, e.g., a iRNA agent having a double stranded region ofless than 40 and preferably less than 30 nucleotides and having one ortwo 1-3 nucleotide single strand 3′ overhangs can be administeredorally.

Oral administration can be in the form of tablets, capsules, gelcapsules, lozenges, troches or liquid syrups. In a preferred embodimentthe composition is applied topically to a surface of the oral cavity.

The invention also provides methods, compositions, and kits for buccaldelivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of iRNA agenthaving a double stranded region of less than 40 and preferably less than30 nucleotides and having one or two 1-3 nucleotide single strand 3′overhangs can be administered to the buccal cavity. The medication canbe sprayed into the buccal cavity or applied directly, e.g., in aliquid, solid, or gel form to a surface in the buccal cavity. Thisadministration is particularly desirable for the treatment ofinflammations of the buccal cavity, e.g., the gums or tongue, e.g., inone embodiment, the buccal administration is by spraying into thecavity, e.g., without inhalation, from a dispenser, e.g., a metered dosespray dispenser that dispenses the pharmaceutical composition and apropellant.

The invention also provides methods, compositions, and kits for oculardelivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAagent described herein, e.g., a sRNA agent having a double strandedregion of less than 40 and preferably less than 30 nucleotides andhaving one or two 1-3 nucleotide single strand 3′ overhangs can beadministered to ocular tissue.

The medications can be applied to the surface of the eye or nearbytissue, e.g., the inside of the eyelid. It can be applied topically,e.g., by spraying, in drops, as an eyewash, or an ointment.Administration can be provided by the subject or by another person,e.g., a health care provider. The medication can be provided in measureddoses or in a dispenser that delivers a metered dose.

The medication can also be administered to the interior of the eye, andcan be introduced by a needle or other delivery device which canintroduce it to a selected area or structure.

Ocular treatment is particularly desirable for treating inflammation ofthe eye or nearby tissue.

The invention also provides methods, compositions, and kits for deliveryof iRNA agents described herein to or through the skin.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAagent described herein, e.g., a sRNA agent having a double strandedregion of less than 40 and preferably less than 30 nucleotides and oneor two 1-3 nucleotide single strand 3′ overhangs can be administereddirectly to the skin.

The medication can be applied topically or delivered in a layer of theskin, e.g., by the use of a microneedle or a battery of microneedleswhich penetrate into the skin, but preferably not into the underlyingmuscle tissue.

In one embodiment, the administration of the iRNA agent composition istopical. In another embodiment, topical administration delivers thecomposition to the dermis or epidermis of a subject. In otherembodiments the topical administration is in the form of transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids or powders. A composition for topical administration can beformulated as a liposome, micelle, emulsion, or other lipophilicmolecular assembly.

In another embodiment, the transdermal administration is applied with atleast one penetration enhancer. In other embodiments, the penetrationcan be enhanced with iontophoresis, phonophoresis, and sonophoresis. Inanother aspect, the invention provides methods, compositions, devices,and kits for pulmonary delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of iRNAagent, e.g., a sRNA agent having a double stranded region of less than40, preferably less than 30 nucleotides and having one or two 1-3nucleotide single strand 3′ overhangs can be administered to thepulmonary system. Pulmonary administration can be achieved by inhalationor by the introduction of a delivery device into the pulmonary system,e.g., by introducing a delivery device which can dispense themedication.

The preferred method of pulmonary delivery is by inhalation. Themedication can be provided in a dispenser which delivers the medication,e.g., wet or dry, in a form sufficiently small such that it can beinhaled. The device can deliver a metered dose of medication. Thesubject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directlyaffect pulmonary tissue, but also for disorders which affect othertissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or aerosol for pulmonary delivery.

In another aspect, the invention provides methods, compositions,devices, and kits for nasal delivery of iRNA agents described herein.Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of iRNAagent, e.g., a sRNA agent having a double stranded region of less than40 and preferably less than 30 nucleotides and having one or two 1-3nucleotide single strand 3′ overhangs can be administered nasally. Nasaladministration can be achieved by introduction of a delivery device intothe nose, e.g., by introducing a delivery device which can dispense themedication.

The preferred method of nasal delivery is by spray, aerosol, liquid,e.g., by drops, of by topical administration to a surface of the nasalcavity. The medication can be provided in a dispenser which delivery ofthe medication, e.g., wet or dry, in a form sufficiently small such thatit can be inhaled. The device can deliver a metered dose of medication.The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affectnasal tissue, but also for disorders which affect other tissue iRNAagents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or for nasal delivery.

In another embodiment, the iRNA agent is packaged in a viral naturalcapsid or in a chemically or enzymatically produced artificial capsid orstructure derived therefrom.

In one aspect, of the invention, the dosage of a pharmaceuticalcomposition including a iRNA agent is administered in order to alleviatethe symptoms of a disease state, e.g., cancer or a cardiovasculardisease.

In another aspect, gene expression in a subject is modulated byadministering a pharmaceutical composition including a iRNA agent. Inother embodiments, a subject is treated with the pharmaceuticalcomposition by any of the methods mentioned above. In anotherembodiment, the subject has cancer.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)composition can be administered as a liposome. For example, thecomposition can be prepared by a method that includes: (1) contacting aiRNA agent with an amphipathic cationic lipid conjugate in the presenceof a detergent; and (2) removing the detergent to form a iRNA agent andcationic lipid complex. In one embodiment, the detergent is cholate,deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS(3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane),novel-ϑ-D-glucopyranoside, lauryl dimethylamine oxide, oroctylglucoside. The iRNA agent can be an sRNA agent. The method caninclude preparing a composition that includes a plurality of iRNAagents, e.g., specific for one or more different endogenous target RNAs.The method can include other features described herein.

In another aspect, a subject is treated by administering a definedamount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent) composition that is in a powdered form. Inone embodiment, the powder is a collection of microparticles. In oneembodiment, the powder is a collection of crystalline particles. Thecomposition can include a plurality of iRNA agents, e.g., specific forone or more different endogenous target RNAs. The method can includeother features described herein.

In one aspect, a subject is treated by administering a defined amount ofa iRNA agent composition that is prepared by a method that includesspray-drying, i.e. atomizing a liquid solution, emulsion, or suspension,immediately exposing the droplets to a drying gas, and collecting theresulting porous powder particles. The composition can include aplurality of iRNA agents, e.g., specific for one or more differentendogenous target RNAs. The method can include other features describedherein.

In one aspect, the iRNA agent, e.g., a double-stranded iRNA agent, orsRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof), isprovided in a powdered, crystallized or other finely divided form, withor without a carrier, e.g., a micro- or nano-particle suitable forinhalation or other pulmonary delivery. In one embodiment, this includesproviding an aerosol preparation, e.g., an aerosolized spray-driedcomposition. The aerosol composition can be provided in and/or dispensedby a metered dose delivery device.

In another aspect, a subject is treated for a condition treatable byinhalation. In one embodiment, this method includes aerosolizing aspray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)composition and inhaling the aerosolized composition. The iRNA agent canbe an sRNA. The composition can include a plurality of iRNA agents,e.g., specific for one or more different endogenous target RNAs. Themethod can include other features described herein.

In another aspect, the invention features a method of treating a subjectthat includes: administering a composition including aneffective/defined amount of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof), wherein the composition is prepared by a method that includesspray-drying, lyophilization, vacuum drying, evaporation, fluid beddrying, or a combination of these techniques

In another aspect, the invention features a method that includes:evaluating a parameter related to the abundance of a transcript in acell of a subject; comparing the evaluated parameter to a referencevalue; and if the evaluated parameter has a preselected relationship tothe reference value (e.g., it is greater), administering a iRNA agent(or a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) tothe subject. In one embodiment, the iRNA agent includes a sequence thatis complementary to the evaluated transcript. For example, the parametercan be a direct measure of transcript levels, a measure of a proteinlevel, a disease or disorder symptom or characterization (e.g., rate ofcell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes:administering a first amount of a composition that comprises an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) to a subject, whereinthe iRNA agent includes a strand substantially complementary to a targetnucleic acid; evaluating an activity associated with a protein encodedby the target nucleic acid; wherein the evaluation is used to determineif a second amount should be administered. In a preferred embodiment themethod includes administering a second amount of the composition,wherein the timing of administration or dosage of the second amount is afunction of the evaluating. The method can include other featuresdescribed herein.

In another aspect, the invention features a method of administering asource of a double-stranded iRNA agent (ds iRNA agent) to a subject. Themethod includes administering or implanting a source of a ds iRNA agent,e.g., a sRNA agent, that (a) includes a double-stranded region that is19-25 nucleotides long, preferably 21-23 nucleotides, (b) iscomplementary to a target RNA (e.g., an endogenous RNA or a pathogenRNA), and, optionally, (c) includes at least one 3′ overhang 1-5 ntlong. In one embodiment, the source releases ds iRNA agent over time,e.g. the source is a controlled or a slow release source, e.g., amicroparticle that gradually releases the ds iRNA agent. In anotherembodiment, the source is a pump, e.g., a pump that includes a sensor ora pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)including a nucleotide sequence complementary to a target RNA, e.g.,substantially and/or exactly complementary. The target RNA can be atranscript of an endogenous human gene. In one embodiment, the iRNAagent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b)is complementary to an endogenous target RNA, and, optionally, (c)includes at least one 3′ overhang 1-5 nt long. In one embodiment, thepharmaceutical composition can be an emulsion, microemulsion, cream,jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agentmixed with a topical delivery agent. The topical delivery agent can be aplurality of microscopic vesicles. The microscopic vesicles can beliposomes. In a preferred embodiment the liposomes are cationicliposomes.

In another aspect, the pharmaceutical composition includes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) admixed with a topicalpenetration enhancer. In one embodiment, the topical penetrationenhancer is a fatty acid. The fatty acid can be arachidonic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt.The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid,glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid,sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable saltthereof.

In another embodiment, the penetration enhancer is a chelating agent.The chelating agent can be EDTA, citric acid, a salicyclate, a N-acylderivative of collagen, laureth-9, an N-amino acyl derivative of abeta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g.,an ionic or nonionic surfactant. The surfactant can be sodium laurylsulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether,a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from agroup consisting of unsaturated cyclic ureas, 1-alkyl-alkones,1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents andmixtures thereof. In yet another embodiment the penetration enhancer canbe a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in aform suitable for oral delivery. In one embodiment, oral delivery can beused to deliver an iRNA agent composition to a cell or a region of thegastro-intestinal tract, e.g., small intestine, colon (e.g., to treat acolon cancer), and so forth. The oral delivery form can be tablets,capsules or gel capsules. In one embodiment, the iRNA agent of thepharmaceutical composition modulates expression of a cellular adhesionprotein, modulates a rate of cellular proliferation, or has biologicalactivity against eukaryotic pathogens or retroviruses. In anotherembodiment, the pharmaceutical composition includes an enteric materialthat substantially prevents dissolution of the tablets, capsules or gelcapsules in a mammalian stomach. In a preferred embodiment the entericmaterial is a coating. The coating can be acetate phthalate, propyleneglycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a penetration enhancer. The penetration enhancercan be a bile salt or a fatty acid. The bile salt can be ursodeoxycholicacid, chenodeoxycholic acid, and salts thereof. The fatty acid can becapric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent and a delivery vehicle. In one embodiment, theiRNA agent is (a) is 19-25 nucleotides long, preferably 21-23nucleotides, (b) is complementary to an endogenous target RNA, and,optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) to a cell by a topical route ofadministration. The delivery vehicle can be microscopic vesicles. In oneexample the microscopic vesicles are liposomes. In a preferredembodiment the liposomes are cationic liposomes. In another example themicroscopic vesicles are micelles.

In one aspect, the invention features a method for making apharmaceutical composition, the method including: (1) contacting an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent) with a amphipathic cationic lipid conjugate in the presence of adetergent; and (2) removing the detergent to form a iRNA agent andcationic lipid complex.

In another aspect, the invention features a pharmaceutical compositionproduced by a method including: (1) contacting an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent) with aamphipathic cationic lipid conjugate in the presence of a detergent; and(2) removing the detergent to form a iRNA agent and cationic lipidcomplex. In one embodiment, the detergent is cholate, deoxycholate,lauryl sarcosine, octanoyl sucrose, CHAPS(3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane),novel-ϑ-D-glucopyranoside, lauryl dimethylamine oxide, oroctylglucoside. In another embodiment, the amphipathic cationic lipidconjugate is biodegradable. In yet another embodiment the pharmaceuticalcomposition includes a targeting ligand.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in aninjectable dosage form. In one embodiment, the injectable dosage form ofthe pharmaceutical composition includes sterile aqueous solutions ordispersions and sterile powders. In a preferred embodiment the sterilesolution can include a diluent such as water; saline solution; fixedoils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) inoral dosage form. In one embodiment, the oral dosage form is selectedfrom the group consisting of tablets, capsules and gel capsules. Inanother embodiment, the pharmaceutical composition includes an entericmaterial that substantially prevents dissolution of the tablets,capsules or gel capsules in a mammalian stomach. In a preferredembodiment the enteric material is a coating. The coating can be acetatephthalate, propylene glycol, sorbitan monoleate, cellulose acetatetrimellitate, hydroxy propyl methyl cellulose phthalate or celluloseacetate phthalate. In one embodiment, the oral dosage form of thepharmaceutical composition includes a penetration enhancer, e.g., apenetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in arectal dosage form. In one embodiment, the rectal dosage form is anenema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in avaginal dosage form. In one embodiment, the vaginal dosage form is asuppository. In another embodiment, the vaginal dosage form is a foam,cream, or gel.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in apulmonary or nasal dosage form. In one embodiment, the iRNA agent isincorporated into a particle, e.g., a macroparticle, e.g., amicrosphere. The particle can be produced by spray drying,lyophilization, evaporation, fluid bed drying, vacuum drying, or acombination thereof. The microsphere can be formulated as a suspension,a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) composition suitable for inhalation by asubject, including: (a) a therapeutically effective amount of a iRNAagent suitable for treating a condition in the subject by inhalation;(b) a pharmaceutically acceptable excipient selected from the groupconsisting of carbohydrates and amino acids; and (c) optionally, adispersibility-enhancing amount of a physiologically-acceptable,water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate canbe selected from the group consisting of monosaccharides, disaccharides,trisaccharides, and polysaccharides. In a preferred embodiment thecarbohydrate is a monosaccharide selected from the group consisting ofdextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. Inanother preferred embodiment the carbohydrate is a disaccharide selectedfrom the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In oneembodiment, the amino acid is a hydrophobic amino acid. In a preferredembodiment the hydrophobic amino acid is selected from the groupconsisting of alanine, isoleucine, leucine, methionine, phenylalanine,proline, tryptophan, and valine. In yet another embodiment the aminoacid is a polar amino acid. In a preferred embodiment the amino acid isselected from the group consisting of arginine, histidine, lysine,cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acidand glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selectedfrom the group consisting of human serum albumin, α-lactalbumin,trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter (MMD) of less than 10 microns.In another embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter of less than 5 microns. In yetanother embodiment the spray-dried iRNA agent composition includesparticles having a mass median aerodynamic diameter (MMAD) of less than5 microns.

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). In certain embodimentsthe individual components of the pharmaceutical formulation may beprovided in one container. Alternatively, it may be desirable to providethe components of the pharmaceutical formulation separately in two ormore containers, e.g., one container for an iRNA agent preparation, andat least another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

In another aspect, the invention features a device, e.g., an implantabledevice, wherein the device can dispense or administer a composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof),e.g., a iRNA agent that silences an endogenous transcript. In oneembodiment, the device is coated with the composition. In anotherembodiment the iRNA agent is disposed within the device. In anotherembodiment, the device includes a mechanism to dispense a unit dose ofthe composition. In other embodiments the device releases thecomposition continuously, e.g., by diffusion. Exemplary devices includestents, catheters, pumps, artificial organs or organ components (e.g.,artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having thestructure or characteristics of a crystal, i.e., particles ofthree-dimensional structure in which the plane faces intersect atdefinite angles and in which there is a regular internal structure. Thecompositions of the invention may have different crystalline forms.Crystalline forms can be prepared by a variety of methods, including,for example, spray drying.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, such that the iRNA agent silencesproduction of protein encoded by the target mRNA. In another embodiment,the iRNA agent is “exactly complementary” to a target RNA, e.g., thetarget RNA and the iRNA agent anneal, preferably to form a hybrid madeexclusively of Watson-Crick basepairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in some embodiments, the iRNAagent specifically discriminates a single-nucleotide difference. In thiscase, the iRNA agent only mediates RNAi if exact complementary is foundin the region (e.g., within 7 nucleotides of) the single-nucleotidedifference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) preferably of length less than 100, 200, 300, or400 nucleotides.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. The materials, methods, andexamples are illustrative only and not intended to be limiting. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, usefulmethods and materials are described below. Other features and advantagesof the invention will be apparent from the accompanying drawings anddescription, and from the claims. The contents of all references,pending patent applications and published patents, cited throughout thisapplication are hereby expressly incorporated by reference. In case ofconflict, the present specification, including definitions, willcontrol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural representation of base pairing inpsuedocomplementary siRNA².

FIG. 2 is a schematic representation of dual targeting siRNAs (SEQ IDNOs 3952, 3953, 3954, and 3955 respectively) designed to target the HCVgenome.

FIG. 3 is a schematic representation of pseudocomplementary,bifunctional siRNAs (SEQ ID NOs 3956, 3957, 3958, and 4087 respectively)designed to target the HCV genome.

FIG. 4 is a general synthetic scheme for incorporation of RRMS monomersinto an oligonucleotide.

FIG. 5 is a table of representative RRMS carriers. Panel 1 showspyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel3 shows piperidine-based RRMSs; panel 4 shows morpholine andpiperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 issuccinate or phosphoramidate and R2 is H or a conjugate ligand.

FIG. 6A is a graph depicting levels of luciferase mRNA in livers ofCMV-Luc mice (Xanogen) following intervenous injection (iv) of buffer orsiRNA into the tail vein. Each bar represents data from one mouse. RNAlevels were quantified by QuantiGene Assay (Genospectra, Inc.; Fremont,Calif.)). The Y axis represents chemiluminescence values in counts persecond (CPS).

FIG. 6B is a graph depicting levels of luciferase mRNA in livers ofCMV-Luc mice (Xanogen). The values are averaged from the data depictedin FIG. 6A.

FIG. 7 is a graph depicting the pharmacokinetics ofcholesterol-conjugated and unconjugated siRNA. The diamonds representthe amount of unconjugated ³³P-labeled siRNA (ALN-3000) in mouse plasmaover time; the squares represent the amount of cholesterol-conjugated³³P-labeled siRNA (ALN-3001) in mouse plasma over time. “L1163” isequivalent to ALN3000; “L1163Chol” is equivalent to ALN-3001.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA aresequence-specific mediators of RNA silencing, e.g., by causing RNAdegradation. While not wishing to be bound by theory, it may be that amolecular signal, which may be merely the specific length of thefragments, present in these 21-23 nt fragments recruits cellular factorsthat mediate RNAi. Described herein are methods for preparing andadministering these 21-23 nt fragments, and other iRNAs agents, andtheir use for specifically inactivating gene function. The use of iRNAsagents (or recombinantly produced or chemically synthesizedoligonucleotides of the same or similar nature) enables the targeting ofspecific mRNAs for silencing in mammalian cells. In addition, longerdsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferonresponse which is frequently deleterious, sRNAs do not trigger theinterferon response, at least not to an extent that is deleterious tothe cell and host. In particular, the length of the iRNA agent strandsin an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g.,sufficiently short to avoid inducing a deleterious interferon response.Thus, the administration of a composition of sRNA agent (e.g.,formulated as described herein) to a mammalian cell can be used tosilence expression of a target gene while circumventing the interferonresponse. Further, use of a discrete species of iRNA agent can be usedto selectively target one allele of a target gene, e.g., in a subjectheterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNAagent that disrupts a component of the interferon response, e.g., doublestranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can betreated with a second iRNA agent that includes a sequence complementaryto a target RNA and that has a length that might otherwise trigger theinterferon response.

In a typical embodiment, the subject is a mammal such as a cow, horse,mouse, rat, dog, pig, goat, or a primate. The subject can be a dairymammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken,turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, thesubject is a human, e.g., a normal individual or an individual that has,is diagnosed with, or is predicted to have a disease or disorder.

Further, because iRNA agent mediated silencing persists for several daysafter administering the iRNA agent composition, in many instances, it ispossible to administer the composition with a frequency of less thanonce per day, or, for some instances, only once for the entiretherapeutic regimen. For example, treatment of some cancer cells may bemediated by a single bolus administration, whereas a chronic viralinfection may require regular administration, e.g., once per week oronce per month.

A number of exemplary routes of delivery are described that can be usedto administer an iRNA agent to a subject. In addition, the iRNA agentcan be formulated according to an exemplary method described herein.

iRNA Agent Structure

Described herein are isolated iRNA agents, e.g., RNA molecules,(double-stranded; single-stranded) that mediate RNAi. The iRNA agentspreferably mediate RNAi with respect to an endogenous gene of a subjector to a gene of a pathogen.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, ornucleoside surrogate, all of which are defined herein (see, e.g., thesection below entitled RNA Agents). While numerous modified RNAs andnucleoside surrogates are described, preferred examples include thosewhich have greater resistance to nuclease degradation than do unmodifiedRNAs. Preferred examples include those which have a 2′ sugarmodification, a modification in a single strand overhang, preferably a3′ single strand overhang, or, particularly if single stranded, a 5′modification which includes one or more phosphate groups or one or moreanalogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which canbe cleaved into an RNA agent which can, down regulate the expression ofa target gene, preferably an endogenous or pathogen target RNA. Whilenot wishing to be bound by theory, an iRNA agent may act by one or moreof a number of mechanisms, including post-transcriptional cleavage of atarget mRNA sometimes referred to in the art as RNAi, orpre-transcriptional or pre-translational mechanisms. An iRNA agent caninclude a single strand or can include more than one strands, e.g., itcan be a double stranded iRNA agent. If the iRNA agent is a singlestrand it is particularly preferred that it include a 5′ modificationwhich includes one or more phosphate groups or one or more analogs of aphosphate group.

The iRNA agent should include a region of sufficient homology to thetarget gene, and be of sufficient length in terms of nucleotides, suchthat the iRNA agent, or a fragment thereof, can mediate down regulationof the target gene. (For ease of exposition the term nucleotide orribonucleotide is sometimes used herein in reference to one or moremonomeric subunits of an RNA agent. It will be understood herein thatthe usage of the term “ribonucleotide” or “nucleotide”, herein can, inthe case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions.) Thus, the iRNA agent is or includes a region which is atleast partially, and in some embodiments fully, complementary to thetarget RNA. It is not necessary that there be perfect complementaritybetween the iRNA agent and the target, but the correspondence must besufficient to enable the iRNA agent, or a cleavage product thereof, todirect sequence specific silencing, e.g., by RNAi cleavage of the targetRNA, e.g., mRNA.

Complementarity, or degree of homology with the target strand, is mostcritical in the antisense strand. While perfect complementarity,particularly in the antisense strand, is often desired some embodimentscan include, particularly in the antisense strand, one or more butpreferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to thetarget RNA). The mismatches, particularly in the antisense strand, aremost tolerated in the terminal regions and if present are preferably ina terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5′ and/or 3′ terminus. The sense strand need only be sufficientlycomplementary with the antisense strand to maintain the over all doublestrand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified orinclude nucleoside surrogates in addition to the RRMS. Single strandedregions of an iRNA agent will often be modified or include nucleosidesurrogates, e.g., the unpaired region or regions of a hairpin structure,e.g., a region which links two complementary regions, can havemodifications or nucleoside surrogates. Modification to stabilize one ormore 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, orto favor the antisense sRNA agent to enter into RISC are also favored.Modifications can include C3 (or C6, C7, C12) amino linkers, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), special biotin orfluorescein reagents that come as phosphoramidites and that have anotherDMT-protected hydroxyl group, allowing multiple couplings during RNAsynthesis.

iRNA agents include: molecules that are long enough to trigger theinterferon response (which can be cleaved by Dicer (Bernstein et al.2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencingcomplex)); and, molecules which are sufficiently short that they do nottrigger the interferon response (which molecules can also be cleaved byDicer and/or enter a RISC), e.g., molecules which are of a size whichallows entry into a RISC, e.g., molecules which resemble Dicer-cleavageproducts. Molecules that are short enough that they do not trigger aninterferon response are termed sRNA agents or shorter iRNA agentsherein. “sRNA agent or shorter iRNA agent” as used herein, refers to aniRNA agent, e.g., a double stranded RNA agent or single strand agent,that is sufficiently short that it does not induce a deleteriousinterferon response in a human cell, e.g., it has a duplexed region ofless than 60 but preferably less than 50, 40, or 30 nucleotide pairs.The sRNA agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an endogenous or pathogen target RNA.

Each strand of an sRNA agent can be equal to or less than 30, 25, 24,23, 22, 21, or 20 nucleotides in length. The strand is preferably atleast 19 nucleotides in length. For example, each strand can be between21 and 25 nucleotides in length. Preferred sRNA agents have a duplexregion of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, andone or more overhangs, preferably one or two 3′ overhangs, of 2-3nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an iRNA agent will preferably have one or more of thefollowing properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agentsection below;

(2) if single stranded it will have a 5′ modification which includes oneor more phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or allof the nucleosides, have an anti sense strand that can present bases (ormodified bases) in the proper three dimensional framework so as to beable to form correct base pairing and form a duplex structure with ahomologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or allof the nucleosides, still have “RNA-like” properties, i.e., it willpossess the overall structural, chemical and physical properties of anRNA molecule, even though not exclusively, or even partly, ofribonucleotide-based content. For example, an iRNA agent can contain,e.g., a sense and/or an antisense strand in which all of the nucleotidesugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. Thisdeoxyribonucleotide-containing agent can still be expected to exhibitRNA-like properties. While not wishing to be bound by theory, theelectronegative fluorine prefers an axial orientation when attached tothe C2′ position of ribose. This spatial preference of fluorine can, inturn, force the sugars to adopt a C_(3′)-endo pucker. This is the samepuckering mode as observed in RNA molecules and gives rise to theRNA-characteristic A-family-type helix. Further, since fluorine is agood hydrogen bond acceptor, it can participate in the same hydrogenbonding interactions with water molecules that are known to stabilizeRNA structures. (Generally, it is preferred that a modified moiety atthe 2′ sugar position will be able to enter into H-bonding which is morecharacteristic of the OH moiety of a ribonucleotide than the H moiety ofa deoxyribonucleotide. A preferred iRNA agent will: exhibit aC_(3′)-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of itssugars; exhibit a C_(3′)-endo pucker in a sufficient amount of itssugars that it can give rise to a the RNA-characteristic A-family-typehelix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which isnot a C_(3′)-endo pucker structure. These limitations are particularlypreferably in the antisense strand;

(5) regardless of the nature of the modification, and even though theRNA agent can contain deoxynucleotides or modified deoxynucleotides,particularly in overhang or other single strand regions, it is preferredthat DNA molecules, or any molecule in which more than 50, 60, or 70% ofthe nucleotides in the molecule, or more than 50, 60, or 70% of thenucleotides in a duplexed region are deoxyribonucleotides, or modifieddeoxyribonucleotides which are deoxy at the 2′ position, are excludedfrom the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand iRNA agents are preferably antisensewith regard to the target molecule. In preferred embodiments singlestrand iRNA agents are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). (These modifications can also be used with theantisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enterthe RISC and participate in RISC mediated cleavage of a target mRNA. Asingle strand iRNA agent is at least 14, and more preferably at least15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferablyless than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17,18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex regionwill preferably be equal to or less than 200, 100, or 50, in length.Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23,and 19 to 21 nucleotides pairs in length. The hairpin will preferablyhave a single strand overhang or terminal unpaired region, preferablythe 3′, and preferably of the antisense side of the hairpin. Preferredoverhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agentwhich includes more than one, and preferably two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal toor at least, 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It should be equal to or less than 200, 100, or 50, nucleotidesin length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to orat least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It should be equal to or less than 200, 100, or 50, nucleotidesin length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21nucleotides in length.

The double strand portion of a double stranded iRNA agent should beequal to or at least, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,29, 40, or 60 nucleotide pairs in length. It should be equal to or lessthan 200, 100, or 50, nucleotides pairs in length. Preferred ranges are15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the ds iRNA agent is sufficiently large that it canbe cleaved by an endogenous molecule, e.g., by Dicer, to produce smallerds iRNA agents, e.g., sRNAs agents

It may be desirable to modify one or both of the antisense and sensestrands of a double strand iRNA agent. In some cases they will have thesame modification or the same class of modification but in other casesthe sense and antisense strand will have different modifications, e.g.,in some cases it is desirable to modify only the sense strand. It may bedesirable to modify only the sense strand, e.g., to inactivate it, e.g.,the sense strand can be modified in order to inactivate the sense strandand prevent formation of an active sRNA/protein or RISC. This can beaccomplished by a modification which prevents 5′-phosphorylation of thesense strand, e.g., by modification with a 5′-O-methyl ribonucleotide(see Nykanen et al., (2001) ATP requirements and small interfering RNAstructure in the RNA interference pathway. Cell 107, 309-321.) Othermodifications which prevent phosphorylation can also be used, e.g.,simply substituting the 5′-OH by H rather than 0-Me. Alternatively, alarge bulky group may be added to the 5′-phosphate turning it into aphosphodiester linkage, though this may be less desirable asphosphodiesterases can cleave such a linkage and release a functionalsRNA 5′-end. Antisense strand modifications include 5′ phosphorylationas well as any of the other 5′ modifications discussed herein,particularly the 5′ modifications discussed above in the section onsingle stranded iRNA molecules.

It is preferred that the sense and antisense strands be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains sense andantisense strands, preferable paired to contain an overhang, e.g., oneor two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNAagents will have single-stranded overhangs, preferably 3′ overhangs, of1 or preferably 2 or 3 nucleotides in length at each end. The overhangscan be the result of one strand being longer than the other, or theresult of two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, mostpreferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe sRNA agent range discussed above. sRNA agents can resemble in lengthand structure the natural Dicer processed products from long dsRNAs.Embodiments in which the two strands of the sRNA agent are linked, e.g.,covalently linked are also included. Hairpin, or other single strandstructures which provide the required double stranded region, andpreferably a 3′ overhang are also within the invention.

The isolated iRNA agents described herein, including ds iRNA agents andsRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., atranscript of a gene that encodes a protein. For convenience, such mRNAis also referred to herein as mRNA to be silenced. Such a gene is alsoreferred to as a target gene. In general, the RNA to be silenced is anendogenous gene or a pathogen gene. In addition, RNAs other than mRNA,e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23nucleotides.

As used herein, “specifically hybridizable” and “complementary” areterms which are used to indicate a sufficient degree of complementaritysuch that stable and specific binding occurs between a compound of theinvention and a target RNA molecule. Specific binding requires asufficient degree of complementarity to avoid non-specific binding ofthe oligomeric compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, or in the caseof in vitro assays, under conditions in which the assays are performed.The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to atarget RNA, e.g., a target mRNA, such that the iRNA agent silencesproduction of protein encoded by the target mRNA. In another embodiment,the iRNA agent is “exactly complementary” (excluding the RRMS containingsubunit(s)) to a target RNA, e.g., the target RNA and the iRNA agentanneal, preferably to form a hybrid made exclusively of Watson-Crickbasepairs in the region of exact complementarity. A “sufficientlycomplementary” target RNA can include an internal region (e.g., of atleast 10 nucleotides) that is exactly complementary to a target RNA.Moreover, in some embodiments, the iRNA agent specifically discriminatesa single-nucleotide difference. In this case, the iRNA agent onlymediates RNAi if exact complementary is found in the region (e.g.,within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acidmolecule (RNA or DNA) preferably of length less than 100, 200, 300, or400 nucleotides.

RNA agents discussed herein include otherwise unmodified RNA as well asRNA which have been modified, e.g., to improve efficacy, and polymers ofnucleoside surrogates. Unmodified RNA refers to a molecule in which thecomponents of the nucleic acid, namely sugars, bases, and phosphatemoieties, are the same or essentially the same as that which occur innature, preferably as occur naturally in the human body. The art hasreferred to rare or unusual, but naturally occurring, RNAs as modifiedRNAs, see, e.g., Limbach et al., (1994) Summary: the modifiednucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare orunusual RNAs, often termed modified RNAs (apparently because the aretypically the result of a post transcriptionally modification) arewithin the term unmodified RNA, as used herein. Modified RNA as usedherein refers to a molecule in which one or more of the components ofthe nucleic acid, namely sugars, bases, and phosphate moieties, aredifferent from that which occur in nature, preferably different fromthat which occurs in the human body. While they are referred to asmodified “RNAs,” they will of course, because of the modification,include molecules which are not RNAs. Nucleoside surrogates aremolecules in which the ribophosphate backbone is replaced with anon-ribophosphate construct that allows the bases to the presented inthe correct spatial relationship such that hybridization issubstantially similar to what is seen with a ribophosphate backbone,e.g., non-charged mimics of the ribophosphate backbone. Examples of allof the above are discussed herein.

Much of the discussion below refers to single strand molecules. In manyembodiments of the invention a double stranded iRNA agent, e.g., apartially double stranded iRNA agent, is required or preferred. Thus, itis understood that that double stranded structures (e.g. where twoseparate molecules are contacted to form the double stranded region orwhere the double stranded region is formed by intramolecular pairing(e.g., a hairpin structure)) made of the single stranded structuresdescribed below are within the invention. Preferred lengths aredescribed elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin a nucleic acid, e.g., a modification of a base, or a phosphatemoiety, or the a non-linking O of a phosphate moiety. In some cases themodification will occur at all of the subject positions in the nucleicacid but in many, and infact in most cases it will not. By way ofexample, a modification may only occur at a 3′ or 5′ terminal position,may only occur in a terminal regions, e.g. at a position on a terminalnucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of an RNA or may only occur in a single strand region of an RNA.E.g., a phosphorothioate modification at a non-linking 0 position mayonly occur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular bases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of aribonucleic acid. The basic components are the ribose sugar, the base,the terminal phosphates, and phosphate internucleotide linkers. Wherethe bases are naturally occurring bases, e.g., adenine, uracil, guanineor cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (asdepicted) and W, X, Y, and Z are all O, Formula 1 represents a naturallyoccurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in someapplications, e.g., unmodified oligoribonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications to one ormore of the above RNA components can confer improved properties, and,e.g., can render oligoribonucleotides more stable to nucleases.Unmodified oligoribonucleotides may also be less than optimal in termsof offering tethering points for attaching ligands or other moieties toan iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or moreof:

(i) alteration, e.g., replacement, of one or both of the non-linking (Xand Y) phosphate oxygens and/or of one or more of the linking (W and Z)phosphate oxygens (When the phosphate is in the terminal position, oneof the positions W or Z will not link the phosphate to an additionalelement in a naturally occurring ribonucleic acid. However, forsimplicity of terminology, except where otherwise noted, the W positionat the 5′ end of a nucleic acid and the terminal Z position at the 3′end of a nucleic acid, are within the term “linking phosphate oxygens”as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesalereplacement of the ribose sugar with a structure other than ribose,e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with“dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone(bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal,modification or replacement of a terminal phosphate group or conjugationof a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′end of RNA.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule.

It is understood that the actual electronic structure of some chemicalentities cannot be adequately represented by only one canonical form(i.e. Lewis structure). While not wishing to be bound by theory, theactual structure can instead be some hybrid or weighted average of twoor more canonical forms, known collectively as resonance forms orstructures. Resonance structures are not discrete chemical entities andexist only on paper. They differ from one another only in the placementor “localization” of the bonding and nonbonding electrons for aparticular chemical entity. It can be possible for one resonancestructure to contribute to a greater extent to the hybrid than theothers. Thus, the written and graphical descriptions of the embodimentsof the present invention are made in terms of what the art recognizes asthe predominant resonance form for a particular species. For example,any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)would be represented by X=O and Y=N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-linking oxygen atoms (i.e., X and Yin Formula 1 above). However, the phosphate group can be modified byreplacing one of the oxygens with a different substituent. One result ofthis modification to RNA phosphate backbones can be increased resistanceof the oligoribonucleotide to nucleolytic breakdown. Thus while notwishing to be bound by theory, it can be desirable in some embodimentsto introduce alterations which result in either an uncharged linker or acharged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. Phosphorodithioates have both non-linking oxygensreplaced by sulfur. Unlike the situation where only one of X or Y isaltered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of oligoribonucleotides diastereomers.Diastereomer formation can result in a preparation in which theindividual diastereomers exhibit varying resistance to nucleases.Further, the hybridization affinity of RNA containing chiral phosphategroups can be lower relative to the corresponding unmodified RNAspecies. Thus, while not wishing to be bound by theory, modifications toboth X and Y which eliminate the chiral center, e.g. phosphorodithioateformation, may be desirable in that they cannot produce diastereomermixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkylor aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linkingoxygen (i.e., W or Z in Formula 1) with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylenephosphonates). The replacement can occur at a terminaloxygen (position W (3′) or position Z (5′). Replacement of W with carbonor Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a 2′alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecularnucleophilic attack on the linker phosphorus atom. Again, while notwishing to be bound by theory, it can be desirable to some embodimentsto introduce alterations in which alkoxide formation at the 2′ positionis not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino functionality. Preferredsubstitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified RNA can include nucleotidescontaining e.g., arabinose, as the sugar.

Modified RNA's can also include “abasic” sugars, which lack a nucleobaseat C-1′. These abasic sugars can also be further contain modificationsat one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used incombination with one or more phosphate linker modifications (e.g.,phosphorothioate). The so-called “chimeric” oligonucleotides are thosethat contain two or more different modifications.

The modification can also entail the wholesale replacement of a ribosestructure with another entity at one or more sites in the iRNA agent.These modifications are described in section entitled RiboseReplacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors (cf. Bracket I in Formula 1 above). While not wishing to bebound by theory, it is believed that since the charged phosphodiestergroup is the reaction center in nucleolytic degradation, its replacementwith neutral structural mimics should impart enhanced nucleasestability. Again, while not wishing to be bound by theory, it can bedesirable, in some embodiment, to introduce alterations in which thecharged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group includesiloxane, carbonate, carboxymethyl, carbamate, amide, thioether,ethylene oxide linker, sulfonate, sulfonamide, thioformacetal,formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.Preferred replacements include the methylenecarbonylamino andmethylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates (see Bracket II of Formula 1 above).While not wishing to be bound by theory, it is believed that the absenceof a repetitively charged backbone diminishes binding to proteins thatrecognize polyanions (e.g. nucleases). Again, while not wishing to bebound by theory, it can be desirable in some embodiment, to introducealterations in which the bases are tethered by a neutral surrogatebackbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptidenucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a spacer. The terminal atom of the spacer canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—,—(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g.,n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotinand fluorescein reagents. When a spacer/phosphate-functional molecularentity-spacer/phosphate array is interposed between two strands of iRNAagents, this array can substitute for a hairpin RNA loop in ahairpin-type RNA agent. The 3′ end can be an —OH group. While notwishing to be bound by theory, it is believed that conjugation ofcertain moieties can improve transport, hybridization, and specificityproperties. Again, while not wishing to be bound by theory, it may bedesirable to introduce terminal alterations that improve nucleaseresistance. Other examples of terminal modifications include dyes,intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene,mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclicaromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol,cholic acid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptideconjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents,phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, includingas discussed elsewhere herein to modulate activity or to modulateresistance to degradation. Terminal modifications useful for modulatingactivity include modification of the 5′ end with phosphate or phosphateanalogs. E.g., in preferred embodiments iRNA agents, especiallyantisense strands, are 5′ phosphorylated or include a phosphoryl analogat the 5′ prime terminus. 5′-phosphate modifications include those whichare compatible with RISC mediated gene silencing. Suitable modificationsinclude: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications useful for increasing resistance to degradationinclude

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases” and “universal bases,” can be employed.Examples include without limitation 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 5-halouracil and cytosine,5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyl adenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, butthey can be useful for other reasons, e.g., some, e.g.,2,6-diaminopurine and 2 amino purine, are fluorescent. Modified basescan reduce target specificity. This should be taken into considerationin the design of iRNA agents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNA's

One can evaluate a candidate RNA agent, e.g., a modified RNA, for aselected property by exposing the agent or modified molecule and acontrol molecule to the appropriate conditions and evaluating for thepresence of the selected property. For example, resistance to adegradent can be evaluated as follows. A candidate modified RNA (andpreferably a control molecule, usually the unmodified form) can beexposed to degradative conditions, e.g., exposed to a milieu, whichincludes a degradative agent, e.g., a nuclease. E.g., one can use abiological sample, e.g., one that is similar to a milieu, which might beencountered, in therapeutic use, e.g., blood or a cellular fraction,e.g., a cell-free homogenate or disrupted cells. The candidate andcontrol could then be evaluated for resistance to degradation by any ofa number of approaches. For example, the candidate and control could belabeled, preferably prior to exposure, with, e.g., a radioactive orenzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control andmodified RNA's can be incubated with the degradative agent, andoptionally a control, e.g., an inactivated, e.g., heat inactivated,degradative agent. A physical parameter, e.g., size, of the modified andcontrol molecules are then determined. They can be determined by aphysical method, e.g., by polyacrylamide gel electrophoresis or a sizingcolumn, to assess whether the molecule has maintained its originallength, or assessed functionally. Alternatively, Northern blot analysiscan be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. Afunctional assay can be applied initially or after an earliernon-functional assay, (e.g., assay for resistance to degradation) todetermine if the modification alters the ability of the molecule tosilence gene expression. For example, a cell, e.g., a mammalian cell,such as a mouse or human cell, can be co-transfected with a plasmidexpressing a fluorescent protein, e.g., GFP, and a candidate RNA agenthomologous to the transcript encoding the fluorescent protein (see,e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFPmRNA can be assayed for the ability to inhibit GFP expression bymonitoring for a decrease in cell fluorescence, as compared to a controlcell, in which the transfection did not include the candidate dsRNA,e.g., controls with no agent added and/or controls with a non-modifiedRNA added. Efficacy of the candidate agent on gene expression can beassessed by comparing cell fluorescence in the presence of the modifiedand unmodified dsRNA agents.

In an alternative functional assay, a candidate dsRNA agent homologousto an endogenous mouse gene, preferably a maternally expressed gene,such as c-mos, can be injected into an immature mouse oocyte to assessthe ability of the agent to inhibit gene expression in vivo (see, e.g.,WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintainarrest in metaphase II, can be monitored as an indicator that the agentis inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNAagent would cause the oocyte to exit metaphase arrest and initiateparthenogenetic development (Colledge et al. Nature 370: 65-68, 1994;Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modifiedagent on target RNA levels can be verified by Northern blot to assay fora decrease in the level of target mRNA, or by Western blot to assay fora decrease in the level of target protein, as compared to a negativecontrol. Controls can include cells in which with no agent is addedand/or cells in which a non-modified RNA is added.

References

General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be with solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter 3,2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4,Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can beused herein.

The disclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or 5,366,878. The preparation of phosphotriesteroligoribonucleotides is described in U.S. Pat. No. 5,023,243. Thepreparation of borano phosphate oligoribonucleotide is described in U.S.Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-aminophosphoramidate oligoribonucleotides is described in U.S. Pat. No.5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides isdescribed in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.Preparation of sulfur bridged nucleotides is described in Sproat et al.Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. TetrahedronLett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Bases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.Additional references can be disclosed in the above section on basemodifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R₁, R², and R³ are each, independently, H,(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine,N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methyl cytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyl adenine,N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine,N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ togethercombine with R⁷ to form an [—O—CH₂—] covalently bound bridge between thesugar 2′ and 4′ carbons.

A¹ is:

H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosenfrom 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′),5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′),5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-)).

A² is:

A³ is:

and

A⁴ is:

H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(nSR) ¹⁰;O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰;O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰,N-Q-R¹⁰, S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻,H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each ofwhich may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH,(CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰;O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰,O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰;NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰,S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰,O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰,N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent describedherein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂-]covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5dyes); sulfur, silicon, boron or ester protecting group; intercalatingagents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatichydrocarbons (e.g., phenazine, dihydrophenazine), artificialendonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid,adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl,cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes,haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin,vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole,bisimidazole, histamine, imidazole clusters, acridine-imidazoleconjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. mis 0-1,000,000, and n is 0-20. Q is a spacer selected from the groupconsisting of abasic sugar, amide, carboxy, oxyamine, oxyimine,thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin orfluorescein reagents.

Preferred RNA agents in which the entire phosphate group has beenreplaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent,in which L is a linker, wherein one or both L may be present or absentand is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g);O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the groupconsisting of siloxane, carbonate, carboxymethyl, carbamate, amide,thioether, ethylene oxide linker, sulfonate, sulfonamide,thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides),adenine, guanine, cytosine and uracil, inosine, thymine, xanthine,hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2,N-6 and 0-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine, dihydrouracil,3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine,5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine, N6,N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyl adenine, N6-isopentyladenine,2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylatedbases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸;O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂;NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸; cyano; mercapto, SR⁷;alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl,alkynyl, each of which may be optionally substituted with halo, hydroxy,oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy,amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido,alkylcarbonyl, acyloxy, cyano, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰together combine with R⁷⁰ to form an [—O—CH₂-] covalently bound bridgebetween the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent describedherein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an[—O—CH₂-] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, aminoacid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (seeFormula 4 below):

SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰  FORMULA 4

S is a nucleoside surrogate selected from the group consisting ofmophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is alinker and is selected from the group consisting of CH₂(CH₂)_(g);N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. Mis an amide bond; sulfonamide; sulfinate; phosphate group; modifiedphosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasicnucleotides), adenine, guanine, cytosine and uracil, inosine, thymine,xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine, 7-deazaadenine, 7-deazaguanine,N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil,N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones,5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil,3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine,N⁴-acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyl adenine, N-methylguanines, orO-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent describedherein; and g is 0-2.

Nuclease Resistant Monomers

In one aspect, the invention features a nuclease resistant monomer, or aan iRNA agent which incorporates a nuclease resistant monomer (NMR),such as those described herein and those described in copending,co-owned U.S. Provisional Application Ser. No. 60/469,612 (AttorneyDocket No. 14174-069P01), filed on May 9, 2003, which is herebyincorporated by reference.

In addition, the invention includes iRNA agents having a NMR and anotherelement described herein. E.g., the invention includes an iRNA agentdescribed herein, e.g., a palindromic iRNA agent, an iRNA agent having anon canonical pairing, an iRNA agent which targets a gene describedherein, e.g., a gene active in the liver, an iRNA agent having anarchitecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates a NMR.

An iRNA agent can include monomers which have been modified so as toinhibit degradation, e.g., by nucleases, e.g., endonucleases orexonucleases, found in the body of a subject. These monomers arereferred to herein as NRM's, or nuclease resistance promoting monomersor modifications. In many cases these modifications will modulate otherproperties of the iRNA agent as well, e.g., the ability to interact witha protein, e.g., a transport protein, e.g., serum albumin, or a memberof the RISC (RNA-induced Silencing Complex), or the ability of the firstand second sequences to form a duplex with one another or to form aduplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed thatmodifications of the sugar, base, and/or phosphate backbone in an iRNAagent can enhance endonuclease and exonuclease resistance, and canenhance interactions with transporter proteins and one or more of thefunctional components of the RISC complex. Preferred modifications arethose that increase exonuclease and endonuclease resistance and thusprolong the halflife of the iRNA agent prior to interaction with theRISC complex, but at the same time do not render the iRNA agentresistant to endonuclease activity in the RISC complex. Again, while notwishing to be bound by any theory, it is believed that placement of themodifications at or near the 3′ and/or 5′ end of antisense strands canresult in iRNA agents that meet the preferred nuclease resistancecriteria delineated above. Again, still while not wishing to be bound byany theory, it is believed that placement of the modifications at e.g.,the middle of a sense strand can result in iRNA agents that arerelatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into anydouble-stranded RNA and RNA-like molecule described herein, e.g., aniRNA agent. An iRNA agent may include a duplex comprising a hybridizedsense and antisense strand, in which the antisense strand and/or thesense strand may include one or more of the modifications describedherein. The anti sense strand may include modifications at the 3′ endand/or the 5′ end and/or at one or more positions that occur 1-6 (e.g.,1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sensestrand may include modifications at the 3′ end and/or the 5′ end and/orat any one of the intervening positions between the two ends of thestrand. The iRNA agent may also include a duplex comprising twohybridized antisense strands. The first and/or the second antisensestrand may include one or more of the modifications described herein.Thus, one and/or both antisense strands may include modifications at the3′ end and/or the 5′ end and/or at one or more positions that occur 1-6(e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand.Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet thepreferred nuclease resistance criteria delineated above can include oneor more of the following chemical and/or stereochemical modifications ofthe sugar, base, and/or phosphate backbone:

(i) chiral (S_(P)) thioates. Thus, preferred NRM's include nucleotidedimers with an enriched or pure for a particular chiral form of amodified phosphate group containing a heteroatom at the nonbridgingposition, e.g., Sp or Rp, at the position X, where this is the positionnormally occupied by the oxygen. The atom at X can also be S, Se, Nr₂,or Br₃. When X is S, enriched or chirally pure Sp linkage is preferred.Enriched means at least 70, 80, 90, 95, or 99% of the preferred form.Such NRM's are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base,and/or the phosphorus atom of a phosphate or modified phosphate backbonemoiety. Thus, preferred NRM's include monomers at the terminal positionderivitized at a cationic group. As the 5′ end of an antisense sequenceshould have a terminal —OH or phosphate group this NRM is preferably notused at the 5′ end of an anti-sense sequence. The group should beattached at a position on the base which minimizes interference with Hbond formation and hybridization, e.g., away form the face whichinteracts with the complementary base on the other strand, e.g, at the5′ position of a pyrimidine or a 7-position of a purine. These arediscussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRM'sinclude Non-phosphate linkages, e.g., a linkage of 4 atoms which confersgreater resistance to cleavage than does a phosphate bond. Examplesinclude 3′ CH2-NCH₃—O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus,preferred NRM's can included these structures;

(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, otherpreferred NRM's include: L nucleosides and dimeric nucleotides derivedfrom L-nucleosides; 2′-5′ phosphate, non-phosphate and modifiedphosphate linkages (e.g., thiophosphates, phosphoramidates andboronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′linkages; monomers having an alpha linkage at the 1′ site on the sugar,e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., atargeting moiety or a conjugated ligand described herein conjugated withthe monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasicmonomer, e.g., an abasic monomer as described herein (e.g., anucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclicaromatic monomer as described herein; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM'sinclude monomers, preferably at the terminal position, e.g., the 5′position, in which one or more atoms of the phosphate group isderivatized with a protecting group, which protecting group or groups,are removed as a result of the action of a component in the subject'sbody, e.g, a carboxyesterase or an enzyme present in the subject's body.E.g., a phosphate prodrug in which a carboxy esterase cleaves theprotected molecule resulting in the production of a thioate anion whichattacks a carbon adjacent to the 0 of a phosphate and resulting in theproduction of an unprotected phosphate.

One or more different NRM modifications can be introduced into an iRNAagent or into a sequence of an iRNA agent. An NRM modification can beused more than once in a sequence or in an iRNA agent. As some NRM'sinterfere with hybridization the total number incorporated, should besuch that acceptable levels of iRNA agent duplex formation aremaintained.

In some embodiments NRM modifications are introduced into the terminalthe cleavage site or in the cleavage region of a sequence (a sensestrand or sequence) which does not target a desired sequence or gene inthe subject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one orboth of the non-linking (X and Y) phosphate oxygens and/or of one ormore of the linking (W and Z) phosphate oxygens. Formula X below depictsa phosphate moiety linking two sugar/sugar surrogate-base moities, SB₁and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in thephosphate backbone moiety (X and Y) can be replaced by any one of thefollowing: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., analkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl,etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodifiedphosphate group is achiral. However, replacement of one of thenon-linking oxygens with one of the above atoms or groups of atomsrenders the phosphorus atom chiral; in other words a phosphorus atom ina phosphate group modified in this way is a stereogenic center. Thestereogenic phosphorus atom can possess either the “R” configuration(herein R_(P)) or the “S” configuration (herein S_(P)). Thus if 60% of apopulation of stereogenic phosphorus atoms have the R_(P) configuration,then the remaining 40% of the population of stereogenic phosphorus atomshave the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which aphosphate non-linking oxygen has been replaced by another atom or groupof atoms, may contain a population of stereogenic phosphorus atoms inwhich at least about 50% of these atoms (e.g., at least about 60% ofthese atoms, at least about 70% of these atoms, at least about 80% ofthese atoms, at least about 90% of these atoms, at least about 95% ofthese atoms, at least about 98% of these atoms, at least about 99% ofthese atoms) have the S_(P) configuration. Alternatively, iRNA agentshaving phosphate groups in which a phosphate non-linking oxygen has beenreplaced by another atom or group of atoms may contain a population ofstereogenic phosphorus atoms in which at least about 50% of these atoms(e.g., at least about 60% of these atoms, at least about 70% of theseatoms, at least about 80% of these atoms, at least about 90% of theseatoms, at least about 95% of these atoms, at least about 98% of theseatoms, at least about 99% of these atoms) have the R_(P) configuration.In other embodiments, the population of stereogenic phosphorus atoms mayhave the S_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the R_(P) configuration. In stillother embodiments, the population of stereogenic phosphorus atoms mayhave the R_(P) configuration and may be substantially free ofstereogenic phosphorus atoms having the S_(P) configuration. As usedherein, the phrase “substantially free of stereogenic phosphorus atomshaving the R_(P) configuration” means that moieties containingstereogenic phosphorus atoms having the R_(P) configuration cannot bedetected by conventional methods known in the art (chiral HPLC, ¹H NMRanalysis using chiral shift reagents, etc.). As used herein, the phrase“substantially free of stereogenic phosphorus atoms having the S_(P)configuration” means that moieties containing stereogenic phosphorusatoms having the S_(P) configuration cannot be detected by conventionalmethods known in the art (chiral HPLC, ¹H NMR analysis using chiralshift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain aphosphorothioate group, i.e., a phosphate groups in which a phosphatenon-linking oxygen has been replaced by a sulfur atom. In an especiallypreferred embodiment, the population of phosphorothioate stereogenicphosphorus atoms may have the S_(P) configuration and be substantiallyfree of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimerse.g., formulas X-1 and X-2. The former can be used to introducephosphorothioate

at the 3′ end of a strand, while the latter can be used to introducethis modification at the 5′ end or at a position that occurs e.g., 1, 2,3, 4, 5, or 6 nucleotides from either end of the strand. In the aboveformulas, Y can be 2-cyanoethoxy, W and Z can be 0, R_(2′) can be, e.g.,a substituent that can impart the C-3 endo configuration to the sugar(e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be anatural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groupsthat can result in phosphorothioate-containing dimers having apopulation of stereogenic phosphorus atoms having essentially only theR_(P) configuration (i.e., being substantially free of the S_(P)configuration) or only the S_(P) configuration (i.e., beingsubstantially free of the R_(P) configuration). Alternatively, dimerscan be prepared having a population of stereogenic phosphorus atoms inwhich about 50% of the atoms have the R_(P) configuration and about 50%of the atoms have the S_(P) configuration. Dimers having stereogenicphosphorus atoms with the R_(P) configuration can be identified andseparated from dimers having stereogenic phosphorus atoms with the S_(P)configuration using e.g., enzymatic degradation and/or conventionalchromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groupsto the sugar, base, and/or the phosphorus atom of a phosphate ormodified phosphate backbone moiety. A cationic group can be attached toany atom capable of substitution on a natural, unusual or universalbase. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphatelinkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphatelinkages which can replace the phosphate group include methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include the methylphosphonate and hydroxylamino groups.

3′-Bridging Thiophosphates and 5′-Bridging Thiophosphates; Locked-RNA,2′-5′ Linkages, Inverted Linkages, α-Nucleosides; Conjugate Groups;Abasic Linkages; and 5′-Phosphonates and 5′-Phosphate Prodrugs

Referring to formula X above, modifications can include replacement ofone of the bridging or linking phosphate oxygens in the phosphatebackbone moiety (W and Z). Unlike the situation where only one of X or Yis altered, the phosphorus center in the phosphorodithioates is achiralwhich precludes the formation of iRNA agents containing a stereogenicphosphorus atom.

Modifications can also include linking two sugars via a phosphate ormodified phosphate group through the 2′ position of a first sugar andthe 5′ position of a second sugar. Also contemplated are invertedlinkages in which both a first and second sugar are eached linkedthrough the respective 3′ positions. Modified RNA's can also include“abasic” sugars, which lack a nucleobase at C-1′. The sugar group canalso contain one or more carbons that possess the oppositestereochemical configuration than that of the corresponding carbon inribose. Thus, a modified iRNA agent can include nucleotides containinge.g., arabinose, as the sugar. In another subset of this modification,the natural, unusual, or universal base may have the α-configuration.Modifications can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g.,P(O)(O⁻)₂—X—C^(5′)-sugar (X=CH2, CF2, CHF and 5′-phosphate prodrugs,e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C^(5′)-sugar. In the latter case, theprodrug groups may be decomposed via reaction first with carboxyesterases. The remaining ethyl thiolate group via intramolecular S_(N)2displacement can depart as episulfide to afford the underivatizedphosphate group.

Modification can also include the addition of conjugating groupsdescribed elsewhere herein, which are preferably attached to an iRNAagent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed onlyat the terminus and others which can go at any position. Generally themodifications that can inhibit hybridization so it is preferably to usethem only in terminal regions, and preferable to not use them at thecleavage site or in the cleavage region of an sequence which targets asubject sequence or gene. The can be used anywhere in a sense sequence,provided that sufficient hybridization between the two sequences of theiRNA agent is maintained. In some embodiments it is desirable to put theNRM at the cleavage site or in the cleavage region of a sequence whichdoes not target a subject sequence or gene, as it can minimizeoff-target silencing.

In addition, an iRNA agent described herein can have an overhang whichdoes not form a duplex structure with the other sequence of the iRNAagent—it is an overhang, but it does hybridize, either with itself, orwith another nucleic acid, other than the other sequence of the iRNAagent.

In most cases, the nuclease-resistance promoting modifications will bedistributed differently depending on whether the sequence will target asequence in the subject (often referred to as an anti-sense sequence) orwill not target a sequence in the subject (often referred to as a sensesequence). If a sequence is to target a sequence in the subject,modifications which interfere with or inhibit endonuclease cleavageshould not be inserted in the region which is subject to RISC mediatedcleavage, e.g., the cleavage site or the cleavage region (As describedin Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated byreference, cleavage of the target occurs about in the middle of a 20 or21 nt guide RNA, or about 10 or 11 nucleotides upstream of the firstnucleotide which is complementary to the guide sequence. As used hereincleavage site refers to the nucleotide on either side of the cleavagesite, on the target or on the iRNA agent strand which hybridizes to it.Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of thecleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., atthe terminal position or with 2, 3, 4, or 5 positions of the terminus,of a sequence which targets or a sequence which does not target asequence in the subject.

An iRNA agent can have a first and a second strand chosen from thefollowing:

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which does not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

An iRNA agent can also target two sequences and can have a first andsecond strand chosen from:

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a first strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a first strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a first strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand) and

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end(5′ end NRM modifications are preferentially not at the terminus butrather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of anantisense strand);

a second strand which targets a sequence and which has an NRMmodification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ endand which has a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end;

a second strand which targets a sequence and which preferably does nothave an an NRM modification at the cleavage site or in the cleavageregion;

a second strand which targets a sequence and which dose not have an NRMmodification at the cleavage site or in the cleavage region and one ormore of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positionsfrom the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4,5, or 6 positions from both the 3′ and the 5′ end (5′ end NRMmodifications are preferentially not at the terminus but rather at aposition 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisensestrand).

Ribose Mimics

In one aspect, the invention features a ribose mimic, or an iRNA agentwhich incorporates a ribose mimic, such as those described herein andthose described in copending co-owned U.S. Provisional Application Ser.No. 60/454,962 (Attorney Docket No. 14174-064P01), filed on Mar. 13,2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a ribose mimicand another element described herein. E.g., the invention includes aniRNA agent described herein, e.g., a palindromic iRNA agent, an iRNAagent having a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., a gene active in the liver, an iRNA agent havingan architecture or structure described herein, an iRNA associated withan amphipathic delivery agent described herein, an iRNA associated witha drug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes asecondary hydroxyl group, which can increase efficacy and/or confernuclease resistance to the agent. Nucleases, e.g., cellular nucleases,can hydrolyze nucleic acid phosphodiester bonds, resulting in partial orcomplete degradation of the nucleic acid. The secondary hydroxy groupconfers nuclease resistance to an iRNA agent by rendering the iRNA agentless prone to nuclease degradation relative to an iRNA which lacks themodification. While not wishing to be bound by theory, it is believedthat the presence of a secondary hydroxyl group on the iRNA agent canact as a structural mimic of a 3′ ribose hydroxyl group, thereby causingit to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attachedto a carbon atom substituted by two other carbons and a hydrogen. Thesecondary hydroxyl group that confers nuclease resistance as describedabove can be part of any acyclic carbon-containing group. The hydroxylmay also be part of any cyclic carbon-containing group, and preferablyone or more of the following conditions is met (1) there is no ribosemoiety between the hydroxyl group and the terminal phosphate group or(2) the hydroxyl group is not on a sugar moiety which is coupled to abase. The hydroxyl group is located at least two bonds (e.g., at leastthree bonds away, at least four bonds away, at least five bonds away, atleast six bonds away, at least seven bonds away, at least eight bondsaway, at least nine bonds away, at least ten bonds away, etc.) from theterminal phosphate group phosphorus of the iRNA agent. In preferredembodiments, there are five intervening bonds between the terminalphosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bondsbetween the terminal phosphate group phosphorus and the secondaryhydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agentdescribed herein. The iRNA agent may be connected directly or indirectly(e.g., through a spacer or linker) to “W” of the phosphate group. Thesespacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—,—(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6),abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether,disulfide, thiourea, sulfonamide, or morpholino, or biotin andfluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified(e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group,W and Z can be independently NH, O, or S; and X and Y can beindependently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxyor amino (including alkylamino, arylamino, etc.). Preferably, W, X and Zare O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl,optionally substituted with hydroxyl, amino, halo, phosphate or sulfateand/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken togetherwith with R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken togetherwith with R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken togetherwith with R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl,amino, halo, phosphate or sulfate and/or may be optionally inserted withN, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken togetherwith with R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogenor a functional group; n and q are each independently 1-100; R₈ isC₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C₁-C₁₀ alkyl, C₆-C₁₀aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsetsof iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connecteddirectly or indirectly through a terminal 3′ or 5′ ribose sugar carbonof the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O andY is S.

In still yet another subset of RNAi agent delivery modules, n is 1, andR₂ and R₆ are taken together to form a ring containing six atoms and R₄and R₅ are taken together to form a ring containing six atoms.Preferably, the ring system is a trans-decalin. For example, the RNAiagent delivery module of this subset can include a compound of Formula(Y-1):

The functional group can be, for example, a targeting group (e.g., asteroid or a carbohydrate), a reporter group (e.g., a fluorophore), or alabel (an isotopically labelled moiety). The targeting group can furtherinclude protein binding agents, endothelial cell targeting groups (e.g.,RGD peptides and mimetics), cancer cell targeting groups (e.g., folateVitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates,polyglutamates, polyaspartates), multivalent mannose (for e.g.,macrophage testing), lactose, galactose, N-acetyl-galactosamine,monoclonal antibodies, glycoproteins, lectins, melanotropin, orthyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. The synthesized compounds can be separatedfrom a reaction mixture and further purified by a method such as columnchromatography, high pressure liquid chromatography, orrecrystallization. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize oneor more characteristics of the iRNA agent. In one aspect, the inventionfeatures a ribose replacement monomer subunit (RRMS), or a an iRNA agentwhich incorporates a RRMS, such as those described herein and thosedescribed in one or more of U.S. Provisional Application Ser. No.60/493,986 (Attorney Docket No. 14174-079P01), filed on Aug. 8, 2003,which is hereby incorporated by reference; U.S. Provisional ApplicationSer. No. 60/494,597 (Attorney Docket No. 14174-080P01), filed on Aug.11, 2003, which is hereby incorporated by reference; U.S. ProvisionalApplication Ser. No. 60/506,341 (Attorney Docket No. 14174-080P02),filed on Sep. 26, 2003, which is hereby incorporated by reference; andin U.S. Provisional Application Ser. No. 60/158,453 (Attorney Docket No.14174-080P03), filed on Nov. 7, 2003, which is hereby incorporated byreference.

In addition, the invention includes iRNA agents having a RRMS andanother element described herein. E.g., the invention includes an iRNAagent described herein, e.g., a palindromic iRNA agent, an iRNA agenthaving a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., a gene active in the liver, an iRNA agent havingan archtecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates a RRMS.

The ribose sugar of one or more ribonucleotide subunits of an iRNA agentcan be replaced with another moiety, e.g., a non-carbohydrate(preferably cyclic) carrier. A ribonucleotide subunit in which theribose sugar of the subunit has been so replaced is referred to hereinas a ribose replacement modification subunit (RRMS). A cyclic carriermay be a carbocyclic ring system, i.e., all ring atoms are carbon atoms,or a heterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The carriers further include (i) at least two “backbone attachmentpoints” and (ii) at least one “tethering attachment point.” A “backboneattachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond available for, and that is suitablefor incorporation of the carrier into the backbone, e.g., the phosphate,or modified phosphate, e.g., sulfur containing, backbone, of aribonucleic acid. A “tethering attachment point” as used herein refersto a constituent ring atom of the cyclic carrier, e.g., a carbon atom ora heteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., aligand, e.g., a targeting or delivery moiety, or a moiety which alters aphysical property, e.g., lipophilicity, of an iRNA agent. Optionally,the selected moiety is connected by an intervening tether to the cycliccarrier. Thus, it will include a functional group, e.g., an amino group,or generally, provide a bond, that is suitable for incorporation ortethering of another chemical entity, e.g., a ligand to the constituentring.

Incorporation of one or more RRMSs described herein into an RNA agent,e.g., an iRNA agent, particularly when tethered to an appropriateentity, can confer one or more new properties to the RNA agent and/oralter, enhance or modulate one or more existing properties in the RNAmolecule. E.g., it can alter one or more of lipophilicity or nucleaseresistance. Incorporation of one or more RRMSs described herein into aniRNA agent can, particularly when the RRMS is tethered to an appropriateentity, modulate, e.g., increase, binding affinity of an iRNA agent to atarget mRNA, change the geometry of the duplex form of the iRNA agent,alter distribution or target the iRNA agent to a particular part of thebody, or modify the interaction with nucleic acid binding proteins(e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agentpreferably comprising a first strand and a second strand, wherein atleast one subunit having a formula (R-1) is incorporated into at leastone of said strands.

Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S,CR⁹R¹⁰, or absent; and Z is CR¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a),OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least oneof R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b)(when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ willinclude R^(a) and one will include R^(b); when the RRMS is internal, twoof R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b)); furtherprovided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b)and (CH₂)_(n)OR^(a) may only be present with OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is C₁-C₆ alkyl; R¹³is hydroxy, C₁-C₄ alkoxy, or halo; and R¹⁴ is NR^(c)R⁷.

R^(a) is:

and

R^(b) is:

Each of A and C is, independently, O or S.

B is OH, O⁻, or

R^(c) is H or C₁-C₆ alkyl; R^(d) is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrrolinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidinescaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a piperazinescaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a morpholinoscaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a decalinscaffold, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹together are C⁶ cycloalkyl.

In other preferred embodiments the ribose is replaced with adecalin/indane scafold and, and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²;and R⁵ and R¹¹ together are C⁵ cycloalkyl.

In other preferred embodiments, the ribose is replaced with ahydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-strandedRNA-like molecule described herein, e.g., an iRNA agent. An iRNA agentmay include a duplex comprising a hybridized sense and antisense strand,in which the antisense strand and/or the sense strand may include one ormore of the RRMSs described herein. An RRMS can be introduced at one ormore points in one or both strands of a double-stranded iRNA agent. AnRRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or5′ end of the sense strand or at near (within 2 or 3 positions of) the3′ end of the antisense strand. In some embodiments it is preferred tonot have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ endof the antisense strand. An RRMS can be internal, and will preferably bepositioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand. In an embodiment, aniRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the3′ end of the antisense strand and at (or within 1, 2, or 3 positionsof) the 3′ end of the sense strand. In an embodiment, an iRNA agent mayhave an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of theantisense strand and an RRMS at the 5′ end of the sense strand, in whichboth ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one oneach strand and are hydrophobic moieties. While not wishing to be boundby theory, it is believed that pairing of the hydrophobic ligands canstabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′end of the sense strand, in which both RRMSs may share the same ligand(e.g., cholic acid) via connection of their individual tethers toseparate positions on the ligand. A ligand shared between two proximalRRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end ofthe sense strand and an RRMS at an internal position of the sensestrand. An iRNA agent may have an RRMS at an internal position of thesense strand; or may have an RRMS at an internal position of theantisense strand; or may have an RRMS at an internal position of thesense strand and an RRMS at an internal position of the antisensestrand.

In preferred embodiments the iRNA agent includes a first and secondsequences, which are preferably two separate molecules as opposed to twosequences located on the same strand, have sufficient complementarity toeach other to hybridize (and thereby form a duplex region), e.g., underphysiological conditions, e.g., under physiological conditions but notin contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such thatthe ds iRNA agent includes a single strand or unpaired region at one orboth ends of the molecule. Thus, a ds iRNA agent contains first andsecond sequences, preferable paired to contain an overhang, e.g., one ortwo 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides.Most embodiments will have a 3′ overhang. Preferred sRNA agents willhave single-stranded overhangs, preferably 3′ overhangs, of 1 orpreferably 2 or 3 nucleotides in length at each end. The overhangs canbe the result of one strand being longer than the other, or the resultof two strands of the same length being staggered. 5′ ends arepreferably phosphorylated.

An RNA agent, e.g., an iRNA agent, containing a preferred, butnonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrierincludes two “backbone attachment points” (hydroxyl groups), a“tethering attachment point,” and a ligand, which is connectedindirectly to the carrier via an intervening tether. The RRMS may be the5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W”groups may be a hydroxyl group, and the other “W” group may be a chainof two or more unmodified or modified ribonucleotides. Alternatively,the RRMS may occupy an internal position, and both “W” groups may be oneor more unmodified or modified ribonucleotides. More than one RRMS maybe present in a RNA molecule, e.g., an iRNA agent.

The modified RNA molecule of formula (R-2) can be obtained usingoligonucleotide synthetic methods known in the art. In a preferredembodiment, the modified RNA molecule of formula (II) can be prepared byincorporating one or more of the corresponding RRMS monomer compounds(RRMS monomers, see, e.g., A, B, and C in FIG. 4) into a growing senseor antisense strand, utilizing, e.g., phosphoramidite or H-phosphonatecoupling strategies.

The RRMS monomers generally include two differently functionalizedhydroxyl groups (OFG¹ and OFG² above), which are linked to the carriermolecule (see A in FIG. 4), and a tethering attachment point. As usedherein, the term “functionalized hydroxyl group” means that the hydroxylproton has been replaced by another substituent. As shown inrepresentative structures B and C, one hydroxyl group (OFG¹) on thecarrier is functionalized with a protecting group (PG). The otherhydroxyl group (OFG²) can be functionalized with either (1) a liquid orsolid phase synthesis support reagent (solid circle) directly orindirectly through a linker, L, as in B, or (2) a phosphorus-containingmoiety, e.g., a phosphoramidite as in C. The tethering attachment pointmay be connected to a hydrogen atom, a tether, or a tethered ligand atthe time that the monomer is incorporated into the growing sense orantisense strand (see R in Scheme 1). Thus, the tethered ligand can be,but need not be attached to the monomer at the time that the monomer isincorporated into the growing strand. In certain embodiments, thetether, the ligand or the tethered ligand may be linked to a “precursor”RRMS after a “precursor” RRMS monomer has been incorporated into thestrand.

The (OFG¹) protecting group may be selected as desired, e.g., from T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991). The protecting group is preferablystable under amidite synthesis conditions, storage conditions, andoligonucleotide synthesis conditions. Hydroxyl groups, —OH, arenucleophilic groups (i.e., Lewis bases), which react through the oxygenwith electrophiles (i.e., Lewis acids). Hydroxyl groups in which thehydrogen has been replaced with a protecting group, e.g., atriarylmethyl group or a trialkylsilyl group, are essentially unreactiveas nucleophiles in displacement reactions. Thus, the protected hydroxylgroup is useful in preventing e.g., homocoupling of compoundsexemplified by structure C during oligonucleotide synthesis. A preferredprotecting group is the dimethoxytrityl group.

When the OFG² in B includes a linker, e.g., a long organic linker,connected to a soluble or insoluble support reagent, solution or solidphase synthesis techniques can be employed to build up a chain ofnatural and/or modified ribonucleotides once OFG¹ is deprotected andfree to react as a nucleophile with another nucleoside or monomercontaining an electrophilic group (e.g., an amidite group).Alternatively, a natural or modified ribonucleotide oroligoribonucleotide chain can be coupled to monomer C via an amiditegroup or H-phosphonate group at OFG². Subsequent to this operation, OFG¹can be deblocked, and the restored nucleophilic hydroxyl group can reactwith another nucleoside or monomer containing an electrophilic group(see FIG. 1). R′ can be substituted or unsubstituted alkyl or alkenyl.In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may aC₁-C₁₀ alkyl group, preferably it is a branched group containing threeor more carbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turncontains a liquid or solid phase synthesis support reagent at the otherlinker terminus. The support reagent can be any support medium that cansupport the monomers described herein. The monomer can be attached to aninsoluble support via a linker, L, which allows the monomer (and thegrowing chain) to be solubilized in the solvent in which the support isplaced. The solubilized, yet immobilized, monomer can react withreagents in the surrounding solvent; unreacted reagents and solubleby-products can be readily washed away from the solid support to whichthe monomer or monomer-derived products is attached. Alternatively, themonomer can be attached to a soluble support moiety, e.g., polyethyleneglycol (PEG) and liquid phase synthesis techniques can be used to buildup the chain. Linker and support medium selection is within skill of theart. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or—C(O)(CH₂)_(q)S—, preferably, it is oxalyl, succinyl or thioglycolyl.Standard control pore glass solid phase synthesis supports can not beused in conjunction with fluoride labile 5′ silyl protecting groupsbecause the glass is degraded by fluoride with a significant reductionin the amount of full-length product. Fluoride-stable polystyrene basedsupports or PEG are preferred.

Preferred carriers have the general formula (R-3) provided below. (Inthat structure preferred backbone attachment points can be chosen fromR¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions arechosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴and R⁹. Preferred tethering attachment points include R⁷; R⁵ or R⁶ whenX is CH₂. The carriers are described below as an entity, which can beincorporated into a strand. Thus, it is understood that the structuresalso encompass the situations wherein one (in the case of a terminalposition) or two (in the case of an internal position) of the attachmentpoints, e.g., R¹ or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), isconnected to the phosphate, or modified phosphate, e.g., sulfurcontaining, backbone. E.g., one of the above-named R groups can be—CH2-, wherein one bond is connected to the carrier and one to abackbone atom, e.g., a linking oxygen or a central phosphorus atom.)

X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰; and Z is CR¹¹R¹² orabsent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or(CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, andR¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyloptionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ togetherare C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is H, a ligand, or C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is Hor C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; R¹⁴ isNR^(c)R^(d); R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, orC₂-C₆ alkenyl; R¹⁶ is C₁-C₁₀ alkyl; and R¹⁷ is a liquid or solid phasesupport reagent.

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—; R^(a) is CAr₃; R^(b) isP(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷; R^(c) is H or C₁-C₆ alkyl; and R^(d)is H or a ligand.

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Zis CR¹¹R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², or X isN(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹², or X is CH₂; Y is CR⁹R¹⁰; Z isCR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or theindane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵and R¹¹ together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ringsystem or the 3-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Yis CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primarycarbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in thefive-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directlyto one of the carbons in the five-membered ring (—OFG² in D). For thepyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² maybe attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may beattached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may begeminally substituted to one of the above-referenced carbons. For the3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 andOFG² may be attached to C-4. The pyrroline- and 3-hydroxyproline-basedmonomers may therefore contain linkages (e.g., carbon-carbon bonds)wherein bond rotation is restricted about that particular linkage, e.g.restriction resulting from the presence of a ring. Thus, CH₂OFG¹ andOFG² may be cis or trans with respect to one another in any of thepairings delineated above Accordingly, all cis/trans isomers areexpressly included. The monomers may also contain one or more asymmetriccenters and thus occur as racemates and racemic mixtures, singleenantiomers, individual diastereomers and diastereomeric mixtures. Allsuch isomeric forms of the monomers are expressly included. Thetethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperidine ringsystem (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group (n=1) or ethylene group (n=2), connected to one of thecarbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² ispreferably attached directly to one of the carbons in the six-memberedring (—OFG² in E). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminalmanner on the ring, i.e., both groups may be attached to the samecarbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ andOFG² may be disposed in a vicinal manner on the ring, i.e., both groupsmay be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ maybe attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ maybe attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹may be attached to C-4 and OFG² may be attached to C-3. Thepiperidine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included. The tethering attachment point is preferablynitrogen.

In certain embodiments, the carrier may be based on the piperazine ringsystem (F), e.g., X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², orthe morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Zis CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., amethylene group, connected to one of the carbons in the six-memberedring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to oneof the carbons in the six-membered rings (—OFG² in F or G). For both Fand G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3;or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminallysubstituted to one of the above-referenced carbons. The piperazine- andmorpholine-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to oneanother in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included. R′″ can be, e.g., C₁-C₆ alkyl, preferably CH₃. Thetethering attachment point is preferably nitrogen in both F and G.

In certain embodiments, the carrier may be based on the decalin ringsystem, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., Xis CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2)connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_(n)OFG¹ in H]. OFG² ispreferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² inH). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on thering, i.e., both groups may be attached to the same carbon, e.g., atC-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may bedisposed in a vicinal manner on the ring, i.e., both groups may beattached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may beattached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may beattached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ maybe attached to C-4 and OFG² may be attached to C-5; or —(CH₂)_(n)OFG¹may be attached to C-5 and OFG² may be attached to C-4. The decalin orindane-based monomers may therefore contain linkages (e.g.,carbon-carbon bonds) wherein bond rotation is restricted about thatparticular linkage, e.g. restriction resulting from the presence of aring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect toone another in any of the pairings delineated above. Accordingly, allcis/trans isomers are expressly included. The monomers may also containone or more asymmetric centers and thus occur as racemates and racemicmixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of the monomers areexpressly included. In a preferred embodiment, the substituents at C-1and C-6 are trans with respect to one another. The tethering attachmentpoint is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus,—(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another.Accordingly, all cis/trans isomers are expressly included. The monomersmay also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers,individual diastereomers and diastereomeric mixtures. All such isomericforms of the monomers are expressly included. The tethering attachmentpoint is preferably nitrogen.

Representative carriers are shown in FIG. 5.

In certain embodiments, a moiety, e.g., a ligand may be connectedindirectly to the carrier via the intermediacy of an intervening tether.Tethers are connected to the carrier at the tethering attachment point(TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g.C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀, C₁-C₆), preferably having at least onenitrogen atom. In preferred embodiments, the nitrogen atom forms part ofa terminal amino group on the tether, which may serve as a connectionpoint for the ligand. Preferred tethers (underlined) includeTAP-(CH₂)_(n)NH₂; TAP-C(O)(CH₂)_(n)NH₂; or TAP-NR″″(CH₂)_(n)NH₂, inwhich n is 1-6 and R″″ is C₁-C₆ alkyl. and R^(d) is hydrogen or aligand. In other embodiments, the nitrogen may form part of a terminaloxyamino group, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether mayoptionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl,and/or optionally inserted with one or more additional heteroatoms,e.g., N, O, or S. Preferred tethered ligands may include, e.g.,TAP-(CH₂)_(n)NH(LIGAND), TAP-C(O)(CH₂)_(n)NH(LIGAND), orTAP-NR″″(CH₂)_(n)NH(LIGAND); TAP-(CH₂)ONH(LIGAND),TAP-C(O)(CH₂ONH(LIGAND), or TAP-NR″″(CH₂)_(n)ONH(LIGAND);TAP-(CH₂)_(n)NHNH₂(LIGAND), TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND), orTAP-NR″″(CH₂)_(n)NHNH₂(LIGAND).

In other embodiments the tether may include an electrophilic moiety,preferably at the terminal position of the tether. Preferredelectrophilic moieties include, e.g., an aldehyde, alkyl halide,mesylate, tosylate, nosylate, or brosylate, or an activated carboxylicacid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferredtethers (underlined) include TAP-(CH₂)_(n)CHO; TAP-C(O)(CH₂)_(n)CHO; orTAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; orTAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)(O)ONHS; orTAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl;TAP-(CH₂)_(n)C(O)OC₆F₅; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅; orTAP-NR″″(CH₂)_(n)C(O)OC₆F₅, in which n is 1-6 and R″″ is C₁-C₆ alkyl; or—(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)CH₂LG, inwhich n is 1-6 and R″″ is C₁-C₆ alkyl (LG can be a leaving group, e.g.,halide, mesylate, tosylate, nosylate, brosylate). Tethering can becarried out by coupling a nucleophilic group of a ligand, e.g., a thiolor amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., tothe carrier of an RRMS. Examples are described below in the context ofan RRMS but that is only preferred, entities can be coupled at otherpoints to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferablycovalently, either directly or indirectly via an intervening tether, tothe RRMS carrier. In preferred embodiments, the ligand is attached tothe carrier via an intervening tether. As discussed above, the ligand ortethered ligand may be present on the RRMS monomer when the RRMS monomeris incorporated into the growing strand. In some embodiments, the ligandmay be incorporated into a “precursor” RRMS after a “precursor” RRMSmonomer has been incorporated into the growing strand. For example, anRRMS monomer having, e.g., an amino-terminated tether (i.e., having noassociated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into agrowing sense or antisense strand. In a subsequent operation, i.e.,after incorporation of the precursor monomer into the strand, a ligandhaving an electrophilic group, e.g., a pentafluorophenyl ester oraldehyde group, can subsequently be attached to the precursor RRMS bycoupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor RRMS tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g, molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a cancer cell,endothelial cell, bone cell. A targeting group can be a thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, Mucincarbohydrate, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose,multivalent fucose, glycosylated polyaminoacids, multivalent galactose,transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid,cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or anRGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g, cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. Preferably, the target tissue is the liver, preferablyparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a seruprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, forexample).

TABLE 1 Exemplary Cell Permeation Peptides Cell Permeation Peptide Aminoacid Sequence Reference Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 6737)Derossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragmentGRKKRRQRRRPPQC (SEQ ID NO: 6738) Vives et al., J. Biol. (48-60) Chem.,272: 16010, 1997 Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al.,Sequence- (SEQ ID NO: 6738) Biochem. Biophys. based peptide Res.Commun., 243: 601, 1998 PVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 6739)Elmquist et al., Exp. Cell Res., 269: 237, 2001 TransportanGWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB (SEQ ID NO: 6740) J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA (SEQ ID Oehlke et al., Mol.model peptide NO: 6741) Ther., 2: 339, 2000 Arg₉ RRRRRRRRR (SEQ ID NO:6742) Mitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial cellKFFKFFKFFK (SEQ ID NO: 6743) wall permeating LL-37LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO: 6744) Cecropin P1SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ ID NO: 6745) α-defensinACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO: 6746) b-defensinDHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO: 6747) BactenecinRKCRIVVIRVCR (SEQ ID NO: 6748) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (SEQ ID NO: 6749) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ IDNO: 6750)

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:6751). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:6752)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:6753)) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:6754))have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered toan iRNA agent via an incorporated monomer unit is a cell targetingpeptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGDmimic. A peptide moiety can range in length from about 5 amino acids toabout 40 amino acids. The peptide moieties can have a structuralmodification, such as to increase stability or direct conformationalproperties. Any of the structural modifications described below can beutilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). The RGD peptide can be linear or cyclic, and can bemodified, e.g., glycosylated or methylated to facilitate targeting tospecific tissues. For example, a glycosylated RGD peptide can deliver aniRNA agent to a tumor cell expressing α_(V)β₃ (Haubner et al., Jour.Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an IA integrin. Thus, onecould use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the I_(v)-ϑ₃ integrinligand. Generally, such ligands can be used to control proliferatingcells and angiogeneis. Preferred conjugates of this type include an iRNAagent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancergene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be anamphipathic α-helical peptide. Exemplary amphipathic α-helical peptidesinclude, but are not limited to, cecropins, lycotoxins, paradaxins,buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins,S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs),magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2Apeptides, Xenopus peptides, esculentinis-1, and caerins. A number offactors will preferably be considered to maintain the integrity of helixstability. For example, a maximum number of helix stabilization residueswill be utilized (e.g., leu, ala, or lys), and a minimum number helixdestabilization residues will be utilized (e.g., proline, or cyclicmonomeric units. The capping residue will be considered (for example Glyis an exemplary N-capping residue and/or C-terminal amidation can beused to provide an extra H-bond to stabilize the helix. Formation ofsalt bridges between residues with opposite charges, separated by i±3,or i±4 positions can provide stability. For example, cationic residuessuch as lysine, arginine, homo-arginine, ornithine or histidine can formsalt bridges with the anionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents can include modified or non-naturally occurring bases, e.g.,bases described in copending and coowned U.S. Provisional ApplicationSer. No. 60/463,772 (Attorney Docket No. 14174-070P01), filed on Apr.17, 2003, which is hereby incorporated by reference and/or in copendingand coowned U.S. Provisional Application Ser. No. 60/465,802 (AttorneyDocket No. 14174-074P01), filed on Apr. 25, 2003, which is herebyincorporated by reference. Monomers and iRNA agents which include suchbases can be made by the methods found in U.S. Provisional ApplicationSer. No. 60/463,772 (Attorney Docket No. 14174-070P01), filed on Apr.17, 2003, and/or in U.S. Provisional Application Ser. No. 60/465,802(Attorney Docket No. 14174-074P01), filed on Apr. 25, 2003.

In addition, the invention includes iRNA agents having a modified ornon-naturally occurring base and another element described herein. E.g.,the invention includes an iRNA agent described herein, e.g., apalindromic iRNA agent, an iRNA agent having a non canonical pairing, aniRNA agent which targets a gene described herein, e.g., a gene active inthe liver, an iRNA agent having an architecture or structure describedherein, an iRNA associated with an amphipathic delivery agent describedherein, an iRNA associated with a drug delivery module described herein,an iRNA agent administered as described herein, or an iRNA agentformulated as described herein, which also incorporates a modified ornon-naturally occurring base.

The synthesis and purification of oligonucleotide peptide conjugates canbe performed by established methods. See, for example, Trufert et al.,Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugatesin Antisense Technology,” in Antisense Drug Technology, ed. S. T.Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified tocreate a constrained peptide that adopts a distinct and specificpreferred conformation, which can increase the potency and selectivityof the peptide. For example, the constrained peptide can be anazapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide issynthesized by replacing the α-carbon of an amino acid with a nitrogenatom without changing the structure of the amino acid side chain. Forexample, the azapeptide can be synthesized by using hydrazine intraditional peptide synthesis coupling methods, such as by reactinghydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be an N-methylpeptide. N-methyl peptides are composed of N-methyl amino acids, whichprovide an additional methyl group in the peptide backbone, therebypotentially providing additional means of resistance to proteolyticcleavage. N-methyl peptides can by synthesized by methods known in theart (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99,2000; Cell Penetrating Peptides: Processes and Applications, Langel,ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate.Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002).For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be a β-peptide.β-peptides form stable secondary structures such as helices, pleatedsheets, turns and hairpins in solutions. Their cyclic derivatives canfold into nanotubes in the solid state. β-peptides are resistant todegradation by proteolytic enzymes. β-peptides can be synthesized bymethods known in the art. For example, an Ant or Tat peptide can be aβ-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate.Oligocarbamate peptides are internalized into a cell by a transportpathway facilitated by carbamate transporters. For example, an Ant orTat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., apeptide or peptidomimetic tethered to an RRMS) can be an oligoureaconjugate (or an oligothiourea conjugate), in which the amide bond of apeptidomimetic is replaced with a urea moiety. Replacement of the amidebond provides increased resistance to degradation by proteolyticenzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In oneembodiment, an oligourea conjugate is tethered to an iRNA agent for usein oral delivery. The backbone in each repeating unit of an oligoureapeptidomimetic can be extended by one carbon atom in comparison with thenatural amino acid. The single carbon atom extension can increasepeptide stability and lipophilicity, for example. An oligourea peptidecan therefore be advantageous when an iRNA agent is directed for passagethrough a bacterial cell wall, or when an iRNA agent must traverse theblood-brain barrier, such as for the treatment of a neurologicaldisorder. In one embodiment, a hydrogen bonding unit is conjugated tothe oligourea peptide, such as to create an increased affinity with areceptor. For example, an Ant or Tat peptide can be an oligoureaconjugate (or an oligothiourea conjugate).

The siRNA peptide conjugates of the invention can be affiliated with,e.g., tethered to, RRMSs occurring at various positions on an iRNAagent. For example, a peptide can be terminally conjugated, on eitherthe sense or the antisense strand, or a peptide can be bisconjugated(one peptide tethered to each end, one conjugated to the sense strand,and one conjugated to the antisense strand). In another option, thepeptide can be internally conjugated, such as in the loop of a shorthairpin iRNA agent. In yet another option, the peptide can be affiliatedwith a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, whichcan encapsulate one or more iRNA agents (such as for delivery to abiological system and/or a cell), and a peptide moiety tethered to theoutside of the carrier molecule, such as for targeting the carriercomplex to a particular tissue or cell type. A carrier complex can carryadditional targeting molecules on the exterior of the complex, orfusogenic agents to aid in cell delivery. The one or more iRNA agentsencapsulated within the carrier can be conjugated to lipophilicmolecules, which can aid in the delivery of the agents to the interiorof the carrier.

A carrier molecule or structure can be, for example, a micelle, aliposome (e.g., a cationic liposome), a nanoparticle, a microsphere, ora biodegradable polymer. A peptide moiety can be tethered to the carriermolecule by a variety of linkages, such as a disulfide linkage, an acidlabile linkage, a peptide-based linkage, an oxyamino linkage or ahydrazine linkage. For example, a peptide-based linkage can be a GFLGpeptide. Certain linkages will have particular advantages, and theadvantages (or disadvantages) can be considered depending on the tissuetarget or intended use. For example, peptide based linkages are stablein the blood stream but are susceptible to enzymatic cleavage in thelysosomes.

Targeting

The iRNA agents of the invention are particularly useful when targetedto the liver. An iRNA agent can be targeted to the liver byincorporation of an RRMS containing a ligand that targets the liver. Forexample, a liver-targeting agent can be a lipophilic moiety. Preferredlipophilic moieties include lipid, cholesterols, oleyl, retinyl, orcholesteryl residues. Other lipophilic moieties that can function asliver-targeting agents include cholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with alow-density lipoprotein (LDL), such as lactosylated LDL. Polymericcarriers complexed with sugar residues can also function to target iRNAagents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/oranalogues thereof, is particularly useful. These agents target, inparticular, the parenchymal cells of the liver. For example, a targetingmoiety can include more than one or preferably two or three galactosemoieties, spaced about 15 angstroms from each other. The targetingmoiety can alternatively be lactose (e.g., three lactose moieties),which is glucose coupled to a galactose. The targeting moiety can alsobe N-Acetyl-Galactosamine, N—Ac-Glucosamine. A mannose ormannose-6-phosphate targeting moiety can be used for macrophagetargeting.

Conjugation of an iRNA agent with a serum albumin (SA), such as humanserum albumin, can also be used to target the iRNA agent to the liver.

An iRNA agent targeted to the liver by an RRMS targeting moietydescribed herein can target a gene expressed in the liver. For example,the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoproteingene, beta-catenin, or c-MET, such as for treating a cancer of theliver. In another embodiment, the iRNA agent can target apoB-100, suchas for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias,e.g., familial combined hyperlipidemia (FCHL), or acquiredhyperlipidemia; hypercholesterolemia; statin-resistanthypercholesterolemia; coronary artery disease (CAD); coronary heartdisease (CHD); or atherosclerosis. In another embodiment, the iRNA agentcan target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon;glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator(PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase;glucose-6-phosphate translocator; glucokinase inhibitory regulatoryprotein; or phosphoenolpyruvate carboxykinase (PEPCK), such as toinhibit hepatic glucose production in a mammal, such as a human, such asfor the treatment of diabetes. In another embodiment, an iRNA agenttargeted to the liver can target Factor V, e.g., the Leiden Factor Vallele, such as to reduce the tendency to form a blood clot. An iRNAagent targeted to the liver can include a sequence which targetshepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). Forexample, an iRNA agent of the invention can target any one of thenonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatmentof hepatitis B, an iRNA agent can target the protein X (HBx) gene, forexample.

Preferred ligands on RRMSs include folic acid, glucose, cholesterol,cholic acid, Vitamin E, Vitamin K, or Vitamin A.

Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers toan alkyl in which one or more hydrogen atoms are replaced by halo, andincludes alkyl moieties in which all hydrogens have been replaced byhalo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may beoptionally inserted with O, N, or S. The terms “aralkyl” refers to analkyl moiety in which an alkyl hydrogen atom is replaced by an arylgroup. Aralkyl includes groups in which more than one hydrogen atom hasbeen replaced by an aryl group. Examples of “aralkyl” include benzyl,9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chaincontaining 2-8 carbon atoms and characterized in having one or moredouble bonds. Examples of a typical alkenyl include, but not limited to,allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term“alkynyl” refers to a straight or branched hydrocarbon chain containing2-8 carbon atoms and characterized in having one or more triple bonds.Some examples of a typical alkynyl are ethynyl, 2-propynyl, and3-methylbutynyl, and propargyl. The sp² and sp³ carbons may optionallyserve as the point of attachment of the alkenyl and alkynyl groups,respectively.

The term “alkoxy” refers to an —O-alkyl radical. The term “aminoalkyl”refers to an alkyl substituted with an amino The term “mercapto” refersto an —SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—,—CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalentspecies of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted by a substituent. Examples of aryl moieties include,but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom capable of substitution can besubstituted by a substituent. The cycloalkyl groups herein described mayalso contain fused rings. Fused rings are rings that share a commoncarbon-carbon bond. Examples of cycloalkyl moieties include, but are notlimited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom capable of substitution can be substituted by asubstituent. The heterocyclyl groups herein described may also containfused rings. Fused rings are rings that share a common carbon-carbonbond. Examples of heterocyclyl include, but are not limited totetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino,pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcapable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” andthe like refer to radicals of adenine, cytosine, guanine, thymine, anduracil.

As used herein, an “unusual” nucleobase can include any one of thefollowing:

-   2-methyladeninyl,-   N6-methyladeninyl,-   2-methylthio-N6-methyladeninyl,-   N6-isopentenyladeninyl,-   2-methylthio-N6-isopentenyladeninyl,-   N6-(cis-hydroxyisopentenyl)adeninyl,-   2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,-   N6-glycinylcarbamoyladeninyl,-   N6-threonylcarbamoyladeninyl,-   2-methylthio-N6-threonyl carbamoyladeninyl,-   N6-methyl-N6-threonylcarbamoyladeninyl,-   N6-hydroxynorvalylcarbamoyladeninyl,-   2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,-   N6,N6-dimethyl adeninyl,-   3-methylcytosinyl,-   5-methylcytosinyl,-   2-thiocytosinyl,-   5-formylcytosinyl,

-   N4-methyl cytosinyl,-   5-hydroxymethylcytosinyl,-   1-methylguaninyl,-   N2-methylguaninyl,-   7-methylguaninyl,-   N2,N2-dimethylguaninyl,

-   N2,N2,7-trimethylguaninyl,-   1-methylguaninyl,-   7-cyano-7-deazaguaninyl,-   7-aminomethyl-7-deazaguaninyl,-   pseudouracilyl,-   dihydrouracilyl,-   5-methyluracilyl,-   1-methylpseudouracilyl,-   2-thiouracilyl,-   4-thiouracilyl,-   2-thiothyminyl-   5-methyl-2-thiouracilyl,-   3-(3-amino-3-carboxypropyl)uracilyl,-   5-hydroxyuracilyl,-   5-methoxyuracilyl,-   uracilyl 5-oxyacetic acid,-   uracilyl 5-oxyacetic acid methyl ester,-   5-(carboxyhydroxymethyl)uracilyl,-   5-(carboxyhydroxymethyl)uracilyl methyl ester,-   5-methoxycarbonylmethyluracilyl,-   5-methoxycarbonylmethyl-2-thiouracilyl,-   5-aminomethyl-2-thiouracilyl,-   5-methylaminomethyluracilyl,-   5-methylaminomethyl-2-thiouracilyl,-   5-methylaminomethyl-2-selenouracilyl,-   5-carbamoylmethyluracilyl,-   5-carboxymethylaminomethyluracilyl,-   5-carboxymethylaminomethyl-2-thiouracilyl,-   3-methyluracilyl,-   1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,-   5-carboxymethyluracilyl,-   5-methyldihydrouracilyl, or-   3-methylpseudouracilyl.

Asymmetrical Modifications

In one aspect, the invention features an iRNA agent which can beasymmetrically modified as described herein.

In addition, the invention includes iRNA agents having asymmetricalmodifications and another element described herein. E.g., the inventionincludes an iRNA agent described herein, e.g., a palindromic iRNA agent,an iRNA agent having a non canonical pairing, an iRNA agent whichtargets a gene described herein, e.g., a gene active in the liver, aniRNA agent having an architecture or structure described herein, an iRNAassociated with an amphipathic delivery agent described herein, an iRNAassociated with a drug delivery module described herein, an iRNA agentadministered as described herein, or an iRNA agent formulated asdescribed herein, which also incorporates an asymmetrical modification.

iRNA agents of the invention can be asymmetrically modified. Anasymmetrically modified iRNA agent is one in which a strand has amodification which is not present on the other strand. An asymmetricalmodification is a modification found on one strand but not on the otherstrand. Any modification, e.g., any modification described herein, canbe present as an asymmetrical modification. An asymmetrical modificationcan confer any of the desired properties associated with a modification,e.g., those properties discussed herein. E.g., an asymmetricalmodification can: confer resistance to degradation, an alteration inhalf life; target the iRNA agent to a particular target, e.g., to aparticular tissue; modulate, e.g., increase or decrease, the affinity ofa strand for its complement or target sequence; or hinder or promotemodification of a terminal moiety, e.g., modification by a kinase orother enzymes involved in the RISC mechanism pathway. The designation ofa modification as having one property does not mean that it has no otherproperty, e.g., a modification referred to as one which promotesstabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanisticmodel, it is believed that asymmetrical modification allows an iRNAagent to be optimized in view of the different or “asymmetrical”functions of the sense and antisense strands. For example, both strandscan be modified to increase nuclease resistance, however, since somechanges can inhibit RISC activity, these changes can be chosen for thesense stand. In addition, since some modifications, e.g., targetingmoieties, can add large bulky groups that, e.g., can interfere with thecleavage activity of the RISC complex, such modifications are preferablyplaced on the sense strand. Thus, targeting moieties, especially bulkyones (e.g. cholesterol), are preferentially added to the sense strand.In one embodiment, an asymmetrical modification in which a phosphate ofthe backbone is substituted with S, e.g., a phosphorothioatemodification, is present in the antisense strand, and a 2′ modification,e.g., 2′ OMe is present in the sense strand. A targeting moiety can bepresent at either (or both) the 5′ or 3′ end of the sense strand of theiRNA agent. In a preferred example, a P of the backbone is replaced withS in the antisense strand, 2′OMe is present in the sense strand, and atargeting moiety is added to either the 5′ or 3′ end of the sense strandof the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has amodification on the sense strand which modification is not found on theantisense strand and the antisense strand has a modification which isnot found on the sense strand.

Each strand can include one or more asymmetrical modifications. By wayof example: one strand can include a first asymmetrical modificationwhich confers a first property on the iRNA agent and the other strandcan have a second asymmetrical modification which confers a secondproperty on the iRNA. E.g., one strand, e.g., the sense strand can havea modification which targets the iRNA agent to a tissue, and the otherstrand, e.g., the antisense strand, has a modification which promoteshybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the sameproperty, e.g., to increase resistance to nucleolytic degradation, butdifferent modifications are chosen for the sense and the antisensestrands, e.g., because the modifications affect other properties aswell. E.g., since some changes can affect RISC activity thesemodifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2′ modification, e.g., a2′ OMe modification, and the other strand has an asymmetricalmodification of the phosphate backbone, e.g., a phosphorothioatemodification. So, in one embodiment the antisense strand has anasymmetrical 2′ OMe modification and the sense strand has anasymmetrical phosphorothioate modification (or vice versa). In aparticularly preferred embodiment the RNAi agent will have asymmetrical2′-O alkyl, preferably, 2′-OMe modifications on the sense strand andasymmetrical backbone P modification, preferably a phosphothioatemodification in the antisense strand. There can be one or multiple2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunitsof the sense strand can be so modified. There can be one or multiplephosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of thesubunits of the antisense strand can be so modified. It is preferable tohave an iRNA agent wherein there are multiple 2′-OMe modifications onthe sense strand and multiple phophorothioate modifications on theantisense strand. All of the subunits on one or both strands can be somodified. A particularly preferred embodiment of multiple asymmetricmodification on both strands has a duplex region about 20-21, andpreferably 19, subunits in length and one or two 3′ overhangs of about 2subunits in length.

Asymmetrical modifications are useful for promoting resistance todegradation by nucleases, e.g., endonucleases. iRNA agents can includeone or more asymmetrical modifications which promote resistance todegradation. In preferred embodiments the modification on the antisensestrand is one which will not interfere with silencing of the target,e.g., one which will not interfere with cleavage of the target. Most ifnot all sites on a strand are vulnerable, to some degree, to degradationby endonucleases. One can determine sites which are relativelyvulnerable and insert asymmetrical modifications which inhibitdegradation. It is often desirable to provide asymmetrical modificationof a UA site in an iRNA agent, and in some cases it is desirable toprovide the UA sequence on both strands with asymmetrical modification.Examples of modifications which inhibit endonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety on the U, especially ona sense strand; modification of the backbone, e.g., with the replacementof an O with an S, in the phosphate backbone, e.g., the provision of aphosphorothioate modification, on the U or the A or both, especially onan antisense strand; replacement of the U with a C5 amino linker;replacement of the A with a G (sequence changes are preferred to belocated on the sense strand and not the antisense strand); andmodification of the at the 2′, 6′, 7′, or 8′ position. Preferredembodiments are those in which one or more of these modifications arepresent on the sense but not the antisense strand, or embodiments wherethe antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation byexonucleases. Asymmetrical modifications can include those in which onlyone strand is modified as well as those in which both are modified. Inpreferred embodiments the modification on the antisense strand is onewhich will not interfere with silencing of the target, e.g., one whichwill not interfere with cleavage of the target. Some embodiments willhave an asymmetrical modification on the sense strand, e.g., in a 3′overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g.,in a 3′ overhang, e.g., at the 3′ terminus. If the modificationsintroduce moieties of different size it is preferable that the larger beon the sense strand. If the modifications introduce moieties ofdifferent charge it is preferable that the one with greater charge be onthe sense strand.

Examples of modifications which inhibit exonucleolytic degradation canbe found herein. Particularly favored modifications include: 2′modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g.,at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule orat the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicatedby the context); modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P in a 3′ overhang, e.g., atthe 3′ terminus; combination of a 2′ modification, e.g., provision of a2′ 0 Me moiety and modification of the backbone, e.g., with thereplacement of a P with an S, e.g., the provision of a phosphorothioatemodification, or the use of a methylated P, in a 3′ overhang, e.g., atthe 3′ terminus; modification with a 3′ alkyl; modification with anabasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus;modification with naproxene, ibuprofen, or other moieties which inhibitdegradation at the 3′ terminus. Preferred embodiments are those in whichone or more of these modifications are present on the sense but not theantisense strand, or embodiments where the antisense strand has fewer ofsuch modifications.

Modifications, e.g., those described herein, which affect targeting canbe provided as asymmetrical modifications. Targeting modifications whichcan inhibit silencing, e.g., by inhibiting cleavage of a target, can beprovided as asymmetrical modifications of the sense strand. Abiodistribution altering moiety, e.g., cholesterol, can be provided inone or more, e.g., two, asymmetrical modifications of the sense strand.Targeting modifications which introduce moieties having a relativelylarge molecular weight, e.g., a molecular weight of more than 400, 500,or 1000 daltons, or which introduce a charged moiety (e.g., having morethan one positive charge or one negative charge) can be placed on thesense strand.

Modifications, e.g., those described herein, which modulate, e.g.,increase or decrease, the affinity of a strand for its compliment ortarget, can be provided as asymmetrical modifications. These include: 5methyl U; 5 methyl C; pseudouridine, Locked nucleic acids, 2 thio U and2-amino-A. In some embodiments one or more of these is provided on theantisense strand.

iRNA agents have a defined structure, with a sense strand and anantisense strand, and in many cases short single strand overhangs, e.g.,of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetricalmodification can be used to optimize the activity of such a structure,e.g., by being placed selectively within the iRNA. E.g., the end regionof the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region caninclude the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang.In preferred embodiments asymmetrical modifications which result in oneor more of the following are used: modifications of the 5′ end of thesense strand which inhibit kinase activation of the sense strand,including, e.g., attachments of conjugates which target the molecule orthe use modifications which protect against 5′ exonucleolyticdegradation; or modifications of either strand, but preferably the sensestrand, which enhance binding between the sense and antisense strand andthereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sensestrand and the 5′ end of the antisense strand is also important forfunction. This region can include the terminal 2, 3, or 4 pairednucleotides and any 3′ overhang. Preferred embodiments includeasymmetrical modifications of either strand, but preferably the sensestrand, which decrease binding between the sense and antisense strandand thereby promote an “open” structure at this end of the molecule.Such modifications include placing conjugates which target the moleculeor modifications which promote nuclease resistance on the sense strandin this region. Modification of the antisense strand which inhibitkinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strandinclude the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S); these modifications can be used topromote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminaland/or internal positions); these modifications can be used to promotenuclease resistance or to enhance binding of the sense to the antisensestrand, the 3′ modifications can be used at the 5′ end of the sensestrand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S)these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the anti sense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);these modifications can be used to promote nuclease resistance or toinhibit binding of the sense to the anti sense strand, or can be used atthe 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleicacids (HNA's) and cyclohexene nucleic acids (CeNA's)); thesemodifications can be used to promote nuclease resistance or to inhibitbinding of the sense to the antisense strand, or can be used at the 5′end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines), thesemodifications can be used to promote nuclease resistance or to enhancebinding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus),these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates;these modifications can be used to promote nuclease resistance or totarget the molecule, or can be used at the 5′ end of the sense strand toavoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisensestrand include the following:

(a) backbone modifications, e.g., modification of a backbone P,including replacement of P with S, or P substituted with alkyl or allyl,e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the3′ end); e.g., with P═O or P═S preferably at the 3′-end, thesemodifications are preferably excluded from the 5′ end region as they mayinterfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe);e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the3′-end, these modifications are preferably excluded from the 5′ endregion as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modificationsare preferably excluded from the 5′ end region as they may contribute tounwanted enhancements of paring between the sense and antisense strands,it is often preferred to have a “loose” structure in the 5′ region,additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

conjugate groups (preferably at terminal positions), e.g., naproxen,biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates,but bulky groups or generally groups which inhibit RISC activity shouldare less preferred.

The 5′-OH of the antisense strand should be kept free to promoteactivity. In some preferred embodiments modifications that promotenuclease resistance should be included at the 3′ end, particularly inthe 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g.,stabilizing, an iRNA agent. The method includes selecting a sequencehaving activity, introducing one or more asymmetric modifications intothe sequence, wherein the introduction of the asymmetric modificationoptimizes a property of the iRNA agent but does not result in a decreasein activity.

The decrease in activity can be less than a preselected level ofdecrease. In preferred embodiments decrease in activity means a decreaseof less than 5, 10, 20, 40, or 50% activity, as compared with anotherwise similar iRNA lacking the introduced modification. Activitycan, e.g., be measured in vivo, or in vitro, with a result in eitherbeing sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and inparticular the properties discussed in the section on asymmetricalmodifications provided herein. The modification can be any asymmetricalmodification, e.g., an asymmetric modification described in the sectionon asymmetrical modifications described herein. Particularly preferredasymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMemodifications, particularly in the sense sequence, and modifications ofa backbone O, particularly phosphorothioate modifications, in theantisense sequence.

In a preferred embodiment a sense sequence is selected and provided withan asymmetrical modification, while in other embodiments an antisensesequence is selected and provided with an asymmetrical modification. Insome embodiments both sense and antisense sequences are selected andeach provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or bothof the sense and antisense sequence. A sequence can have at least 2, 4,6, 8, or more modifications and all or substantially all of the monomersof a sequence can be modified.

TABLE 2 Some examples of Asymmetric Modification This table showsexamples having strand I with a selected modification and strand II witha selected modification. Strand I Strand II Nuclease Resistance (e.g.,2′-OMe) Biodistribution (e.g., P═S) Biodistribution conjugate ProteinBinding Functionality (e.g., Lipophile) (e.g., Naproxen) TissueDistribution Functionality Cell Targeting Functionality (e.g.,Carbohydrates) (e.g., Folate for cancer cells) Tissue DistributionFunctionality Fusogenic Functionality (e.g., Kidney CellTargetingmoieties) (e.g., Polyethylene imines) Cancer Cell TargetingFusogenic Functionality (e.g., RGD peptides and imines) (e.g., peptides)Nuclease Resistance (e.g., 2′-OMe) Increase in binding Affinity (5-Me-C,5-Me-U, 2-thio-U, 2-amino-A, G-clamp, LNA) Tissue DistributionFunctionality RISC activity improving Functionality Helical conformationchanging Tissue Distribution Functionality Functionalities (P═S;lipophile, carbohydrates)

Z—X—Y Architecture

In one aspect, the invention features an iRNA agent which can have aZ—X—Y architecture or structure such as those described herein and thosedescribed in copending, co-owned U.S. Provisional Application Ser. No.60/510,246 (Attorney Docket No. 14174-079P02), filed on Oct. 9, 2003,which is hereby incorporated by reference, and in copending, co-ownedU.S. Provisional Application Ser. No. 60/510,318 (Attorney Docket No.14174-079P03), filed on Oct. 10, 2003, which is hereby incorporated byreference.

In addition, the invention includes iRNA agents having a Z—X—Y structureand another element described herein. E.g., the invention includes aniRNA agent described herein, e.g., a palindromic iRNA agent, an iRNAagent having a non canonical pairing, an iRNA agent which targets a genedescribed herein, e.g., a gene active in the liver, an iRNA associatedwith an amphipathic delivery agent described herein, an iRNA associatedwith a drug delivery module described herein, an iRNA agent administeredas described herein, or an iRNA agent formulated as described herein,which also incorporates a Z—X—Y architecture.

The invention provides an iRNA agent having a first segment, the Zregion, a second segment, the X region, and optionally a third region,the Y region:

Z—X—Y

It may be desirable to modify subunits in one or both of Zand/or Y onone hand and X on the other hand. In some cases they will have the samemodification or the same class of modification but it will more often bethe case that the modifications made in Z and/or Y will differ fromthose made in X.

The Z region typically includes a terminus of an iRNA agent. The lengthof the Z region can vary, but will typically be from 2-14, morepreferably 2-10, subunits in length. It typically is single stranded,i.e., it will not base pair with bases of another strand, though it mayin some embodiments self associate, e.g., to form a loop structure. Suchstructures can be formed by the end of a strand looping back and formingan intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairscan form, having a looped out or connecting region, typically of 2 ormore subunits which do not pair. This can occur at one or both ends of astrand. A typical embodiment of a Z region is a single strand overhang,e.g., an over hang of the length described elsewhere herein. The Zregion can thus be or include a 3′ or 5′ terminal single strand. It canbe sense or antisense strand but if it is antisense it is preferred thatit is a 3-overhang. Typical inter-subunit bonds in the Z region include:P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B)inter-subunit bonds can also be present. (These inter-subunit bonds arediscussed in more detail elsewhere herein.) Other preferred Z regionsubunit modifications (also discussed elsewhere herein) can include:3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alphaconfiguration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a singlestrand iRNA agent, with a corresponding region of the single strand, orin the case of a double stranded iRNA agent, with the correspondingregion of the other strand. The length of the X region can vary but willtypically be between 10-45 and more preferably between 15 and 35subunits. Particularly preferred region X's will include 17, 18, 19, 29,21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengthsare described elsewhere herein and can be used. Typical X regionsubunits include 2′-OH subunits. In typical embodiments phosphateinter-subunit bonds are preferred while phophorothioate or non-phosphatebonds are absent. Other modifications preferred in the X region include:modifications to improve binding, e.g., nucleobase modifications;cationic nucleobase modifications; and C-5 modified pyrimidines, e.g.,allylamines. Some embodiments have 4 or more consecutive 2′OH subunits.While the use of phosphorothioate is sometimes non preferred they can beused if they connect less than 4 consecutive 2′OH subunits.

The Y region will generally conform to the the parameters set out forthe Z regions. However, the X and Z regions need not be the same,different types and numbers of modifications can be present, and infact,one will usually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z regioneach having ribonucleosides in which the 2′-OH is substituted, e.g.,with 2′-OMe or other alkyl; and an X region that includes at least fourconsecutive ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agentcan be modified, e.g., to promote resistance to degradation. Numerousexamples of such modifications are disclosed herein, one example ofwhich is the phosphorothioate linkage. These modifications can beprovided between the subunits of any of the regions, Y, X, and Z.However, it is preferred that their occurrence is minimized and inparticular it is preferred that consecutive modified linkages beavoided.

In a preferred embodiment the iRNA agent will have a Y and Z region eachhaving ribonucleosides in which the 2′-OH is substituted, e.g., with2′-OMe; and an X region that includes at least four consecutivesubunits, e.g., ribonucleoside subunits in which the 2′-OH remainsunsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can bemodified, e.g., to promote resistance to degradation. Thesemodifications can be provided between the subunits of any of theregions, Y, X, and Z. However, it is preferred that they are minimizedand in particular it is preferred that consecutive modified linkages beavoided.

Thus, in a preferred embodiment, not all of the subunit linkages of theiRNA agent are modified and more preferably the maximum number ofconsecutive subunits linked by other than a phospodiester bond will be2, 3, or 4. Particularly preferred iRNA agents will not have four ormore consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunits is joined by modified linkages—i.e. linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X.Thus, in preferred embodiments each of the nucleoside subunit linkagesin X will be phosphodiester linkages, or if subunit linkages in region Xare modified, such modifications will be minimized. E.g., although the Yand/or Z regions can include inter subunit linkages which have beenstabilized against degradation, such modifications will be minimized inthe X region, and in particular consecutive modifications will beminimized. Thus, in preferred embodiments the maximum number ofconsecutive subunits linked by other than a phospodiester bond will be2, 3, or 4. Particularly preferred X regions will not have four or moreconsecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, inwhich each subunits is joined by modified linkages—i.e. linkages thathave been modified to stabilize them from degradation as compared to thephosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and/or Z will be free of phosphorothioatelinkages, though either or both may contain other modifications, e.g.,other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identical 2′moieties.

In a preferred embodiment region X, or in some cases, the entire iRNAagent, has no more than 3 or no more than 4 subunits having identicalsubunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (orother modifications of the subunit linkage) are present in Y and/or Z,but such modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only onephosphorothioate linkage between adjacent subunits, e.g., nucleosides,having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is asecond set of adjacent subunits, e.g., nucleosides, having a 2′modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that secondset is connected by a linkage other than a phosphorothioate linkage,e.g., a modified linkage other than a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has more thanone phosphorothioate linkage connecting adjacent pairs of subunits,e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkylnucleosides, but at least one pair of adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, arebe connected by a linkage other than a phosphorothioate linkage, e.g., amodified linkage other than a phosphorothioate linkage.

In a preferred embodiment one of the above recited limitation onadjacent subunits in Y and or Z is combined with a limitation on thesubunits in X. E.g., one or more phosphorothioate linkages (or othermodifications of the subunit linkage) are present in Y and/or Z, butsuch modified linkages do not connect two adjacent subunits, e.g.,nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g.,any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by alinkage other than a a phosporothioate linkage. In addition, the Xregion has no more than 3 or no more than 4 identical subunits, e.g.,subunits having identical 2′ moieties or the X region has no more than 3or no more than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4of a modification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunits can be present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively. In a preferred embodimentthe frequency of the modification will differ between Y and/or Z and X,e.g., the modification will be present one of Y and/or Z or X and absentin the other.

An X region can include at least one, and preferably 2, 3 or 4 of amodification disclosed herein. Such modifications can be chosen,independently, from any modification described herein, e.g., fromnuclease resistant subunits, subunits with modified bases, subunits withmodified intersubunit linkages, subunits with modified sugars, andsubunits linked to another moiety, e.g., a targeting moiety. In apreferred embodiment more than 1 of such subunits can b present but insome embodiments it is preferred that no more than 1, 2, 3, or 4 of suchmodifications occur, or occur consecutively.

An RRMS (described elsewhere herein) can be introduced at one or morepoints in one or both strands of a double-stranded iRNA agent. An RRMScan be placed in a Y and/or Z region, at or near (within 1, 2, or 3positions) of the 3′ or 5′ end of the sense strand or at near (within 2or 3 positions of) the 3′ end of the antisense strand. In someembodiments it is preferred to not have an RRMS at or near (within 1, 2,or 3 positions of) the 5′ end of the antisense strand. An RRMS can bepositioned in the X region, and will preferably be positioned in thesense strand or in an area of the antisense strand not critical forantisense binding to the target.

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can havedifferential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and anotherelement described herein. E.g., the invention includes an iRNA agentdescribed herein, e.g., a palindromic iRNA agent, an iRNA agent having anon canonical pairing, an iRNA agent which targets a gene describedherein, e.g., a gene active in the liver, an iRNA agent having anarchitecture or structure described herein, an iRNA associated with anamphipathic delivery agent described herein, an iRNA associated with adrug delivery module described herein, an iRNA agent administered asdescribed herein, or an iRNA agent formulated as described herein, whichalso incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplexto disassociate or melt (decreasing the free energy of duplexassociation), in the region of the 5′ end of the antisense strandduplex. This can be accomplished, e.g., by the inclusion of subunitswhich increase the propensity of the duplex to disassociate or melt inthe region of the 5′ end of the antisense strand. It can also beaccomplished by the attachment of a ligand that increases the propensityof the duplex to disassociate of melt in the region of the 5′ end. Whilenot wishing to be bound by theory, the effect may be due to promotingthe effect of an enzyme such as helicase, for example, promoting theeffect of the enzyme in the proximity of the 5′ end of the antisensestrand.

The inventors have also discovered that iRNA agents can be optimized bydecreasing the propensity of the duplex to disassociate or melt(increasing the free energy of duplex association), in the region of the3′ end of the antisense strand duplex. This can be accomplished, e.g.,by the inclusion of subunits which decrease the propensity of the duplexto disassociate or melt in the region of the 3′ end of the antisensestrand. It can also be accomplished by the attachment of ligand thatdecreases the propensity of the duplex to disassociate of melt in theregion of the 5′ end.

Modifications which increase the tendency of the 5′ end of the duplex todissociate can be used alone or in combination with other modificationsdescribed herein, e.g., with modifications which decrease the tendencyof the 3′ end of the duplex to dissociate. Likewise, modifications whichdecrease the tendency of the 3′ end of the duplex to dissociate can beused alone or in combination with other modifications described herein,e.g., with modifications which increase the tendency of the 5′ end ofthe duplex to dissociate.

Decreasing the Stability of the AS 5′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotedissociation or melting (e.g., on the free energy of association ordissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C;

G:U is preferred over G:C;

I:C is preferred over G:C (I=inosine);

mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings;

pairings which include a universal base are preferred over canonicalpairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R₁N₁N₂N₃N₄N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3′ AS 3′ R₃N₁N₂N₃N₄N₅ [N]N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5′ S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵ P⁻⁴ P⁻³ P⁻² P⁻¹ 5′

S indicates the sense strand; AS indicates antisense strand; R₁indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂indicates an optional (though preferred) 3′ sense overhang; R₃ indicatesan optional (though preferred) 3′ antisense sense overhang; R₄ indicatesan optional (and nonpreferred) 5′ antisense overhang; N indicatessubunits; [N] indicates that additional subunit pairs may be present;and P_(x), indicates a paring of sense N_(x) and antisense N. Overhangsare not shown in the P diagram. In some embodiments a 3′ AS overhangcorresponds to region Z, the duplex region corresponds to region X, andthe 3′ S strand overhang corresponds to region Y, as described elsewhereherein. (The diagram is not meant to imply maximum or minimum lengths,on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 5′end of the AS strand. The terminal pair (the most 5′ pair in terms ofthe AS strand) is designated as P⁻¹, and the subsequent pairingpositions (going in the 3′ direction in terms of the AS strand) in theduplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferredregion in which to modify to modulate duplex formation is at P⁻⁵ throughP⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹.Modification at P⁻¹, is particularly preferred, alone or withmodification(s) other position(s), e.g., any of the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions be chosenindependently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairingsor pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve apairing which promotes dissociation will be made in the sense strand,though in some embodiments the change will be made in the anti sensestrand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote dissociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are mismatched pairs, e.g., non-canonical or other thancanonical pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promotestability and inhibit dissociation or melting (e.g., on the free energyof association or dissociation of a particular pairing, the simplestapproach is to examine the pairs on an individual pair basis, thoughnext neighbor or similar analysis can also be used). In terms ofpromoting duplex stability:

G:C is preferred over A:U

Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical orother than canonical pairings

analogs that increase stability are preferred over Watson-Crick matches(A:T, A:U, G:C)

2-amino-A:U is preferred over A:U

2-thio U or 5 Me-thio-U:A are preferred over U:A

G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G

guanadinium-G-clamp:G is preferred over C:G

psuedo uridine:A is preferred over U:A

sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA,which enhance binding are preferred over non-modified moieties and canbe present on one or both strands to enhance stability of the duplex. Itis preferred that pairings which increase the propensity to form aduplex are used at 1 or more of the positions in the duplex at the 3′end of the AS strand. The terminal pair (the most 3′ pair in terms ofthe AS strand) is designated as P₁, and the subsequent pairing positions(going in the 5′ direction in terms of the AS strand) in the duplex aredesignated, P₂, P₃, P₄, P₅, and so on. The preferred region in which tomodify to modulate duplex formation is at P₅ through P₁, more preferablyP₄ through P₁, more preferably P₃ through P₁. Modification at P₁, isparticularly preferred, alone or with modification(s) at otherposition(s), e.g., any of the positions just identified. It is preferredthat at least 1, and more preferably 2, 3, 4, or 5 of the pairs of therecited regions be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crickmatches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹,through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair having an analog that increases stability overWatson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁,through P₄, are a pair in which one or both subunits has a sugarmodification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, whichenhances binding.

G-clamps and guanidinium G-clamps are discussed in the followingreferences: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-ClampContaining Oligonucleotides and Their Inhibition of the HIV-1 Tat-TARInteraction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262,2003; Holmes et al., “Steric inhibition of human immunodeficiency virustype-1 Tat-dependent trans-activation in vitro and in cells byoligonucleotides containing 2′-O-methyl G-clamp ribonucleosideanalogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al.,“Structural basis for recognition of guanosine by a synthetic tricycliccytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta,86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic AcidOligomers Containing Tricyclic Cytosine Analogues,” Organic Letters,4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- andGuanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002;Maier et al., “Nuclease resistance of oligonucleotides containing thetricyclic cytosine analogues phenoxazine and9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nucleaseresistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al.,“A cytosine analog that confers enhanced potency to antisenseoligonucleotides,” Proceedings Of The National Academy Of Sciences OfThe United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′ End of the Duplexand Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decreasethe stability of the AS 5′ end of the duplex and increase the stabilityof the AS 3′ end of the duplex. This can be effected by combining one ormore of the stability decreasing modifications in the AS 5′ end of theduplex with one or more of the stability increasing modifications in theAS 3′ end of the duplex. Accordingly a preferred embodiment includesmodification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ andmore preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularlypreferred, alone or with other position, e.g., the positions justidentified. It is preferred that at least 1, and more preferably 2, 3,4, or 5 of the pairs of one of the recited regions of the AS 5′ end ofthe duplex region be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairingswhich include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and morepreferably P₃ through P₁. Modification at P₁, is particularly preferred,alone or with other position, e.g., the positions just identified. It ispreferred that at least 1, and more preferably 2, 3, 4, or 5 of thepairs of one of the recited regions of the AS 3′ end of the duplexregion be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crickmatches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iRNA agentshaving DMTDS. E.g., when screening a target sequence for candidatesequences for use as iRNA agents one can select sequences having a DMTDSproperty described herein or one which can be modified, preferably withas few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence,and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P inP5 through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., tosilence any gene disclosed herein, to treat any disorder describedherein, in any formulation described herein, and generally in and/orwith the methods and compositions described elsewhere herein. DMTDS iRNAagents can incorporate other modifications described herein, e.g., theattachment of targeting agents or the inclusion of modifications whichenhance stability, e.g., the inclusion of nuclease resistant monomers orthe inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or3'S strand overhangs) which self associate to form intrastrand duplexstructure.

Preferably these iRNA agents will have an architecture described herein.

OTHER EMBODIMENTS

In Vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer forthe efficient delivery of the iRNA agent to a subject, e.g., a mammal,such as a human. The iRNA agent can, for example, be complexed withcyclodextrin. Cyclodextrins have been used as delivery vehicles oftherapeutic compounds. Cyclodextrins can form inclusion complexes withdrugs that are able to fit into the hydrophobic cavity of thecyclodextrin. In other examples, cyclodextrins form non-covalentassociations with other biologically active molecules such asoligonucleotides and derivatives thereof. The use of cyclodextrinscreates a water-soluble drug delivery complex, that can be modified withtargeting or other functional groups. Cyclodextrin cellular deliverysystem for oligonucleotides described in U.S. Pat. No. 5,691,316, whichis hereby incorporated by reference, are suitable for use in methods ofthe invention. In this system, an oligonucleotide is noncovalentlycomplexed with a cyclodextrin, or the oligonucleotide is covalentlybound to adamantine which in turn is noncovalently associated with acyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or alinear oxidized cyclodextrin copolymer having at least one ligand boundto the cyclodextrin copolymer. Delivery systems, as described in U.S.Pat. No. 6,509,323, herein incorporated by reference, are suitable foruse in methods of the invention. An iRNA agent can be bound to thelinear cyclodextrin copolymer and/or a linear oxidized cyclodextrincopolymer. Either or both of the cyclodextrin or oxidized cyclodextrincopolymers can be crosslinked to another polymer and/or bound to aligand.

A composition for iRNA delivery can employ an “inclusion complex,” amolecular compound having the characteristic structure of an adduct. Inthis structure, the “host molecule” spatially encloses at least part ofanother compound in the delivery vehicle. The enclosed compound (the“guest molecule”) is situated in the cavity of the host molecule withoutaffecting the framework structure of the host. A “host” is preferablycyclodextrin, but can be any of the molecules suggested in U.S. PatentPubl. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecularspecies, and the resulting inclusion compounds belong to the class of“host-guest” complexes. Within the host-guest relationship, the bindingsites of the host and guest molecules should be complementary in thestereoelectronic sense. A composition of the invention can contain atleast one polymer and at least one therapeutic agent, generally in theform of a particulate composite of the polymer and therapeutic agent,e.g., the iRNA agent. The iRNA agent can contain one or more complexingagents. At least one polymer of the particulate composite can interactwith the complexing agent in a host-guest or a guest-host interaction toform an inclusion complex between the polymer and the complexing agent.The polymer and, more particularly, the complexing agent can be used tointroduce functionality into the composition. For example, at least onepolymer of the particulate composite has host functionality and forms aninclusion complex with a complexing agent having guest functionality.Alternatively, at least one polymer of the particulate composite hasguest functionality and forms an inclusion complex with a complexingagent having host functionality. A polymer of the particulate compositecan also contain both host and guest functionalities and form inclusioncomplexes with guest complexing agents and host complexing agents. Apolymer with functionality can, for example, facilitate cell targetingand/or cell contact (e.g., targeting or contact to a liver cell),intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the iRNA agent may or may notretain its biological or therapeutic activity. Upon release from thetherapeutic composition, specifically, from the polymer of theparticulate composite, the activity of the iRNA agent is restored.Accordingly, the particulate composite advantageously affords the iRNAagent protection against loss of activity due to, for example,degradation and offers enhanced bioavailability. Thus, a composition maybe used to provide stability, particularly storage or solutionstability, to an iRNA agent or any active chemical compound. The iRNAagent may be further modified with a ligand prior to or afterparticulate composite or therapeutic composition formation. The ligandcan provide further functionality. For example, the ligand can be atargeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determiningtherapeutic toxicity is made easier by the complementarity of the iRNAagent with both a human and a non-human animal sequence. By thesemethods, an iRNA agent can consist of a sequence that is fullycomplementary to a nucleic acid sequence from a human and a nucleic acidsequence from at least one non-human animal, e.g., a non-human mammal,such as a rodent, ruminant or primate. For example, the non-human mammalcan be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus,Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence ofthe iRNA agent could be complementary to sequences within homologousgenes, e.g., oncogenes or tumor suppressor genes, of the non-humanmammal and the human. By determining the toxicity of the iRNA agent inthe non-human mammal, one can extrapolate the toxicity of the iRNA agentin a human. For a more strenuous toxicity test, the iRNA agent can becomplementary to a human and more than one, e.g., two or three or more,non-human animals.

The methods described herein can be used to correlate any physiologicaleffect of an iRNA agent on a human, e.g., any unwanted effect, such as atoxic effect, or any positive, or desired effect.

Delivery Module

In one aspect, the invention features a drug delivery conjugate ormodule, such as those described herein and those described in copending,co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar.12, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents described herein, e.g.,a palindromic iRNA agent, an iRNA agent having a non canonical pairing,an iRNA agent which targets a gene described herein, e.g., a gene activein the liver, an iRNA agent having a chemical modification describedherein, e.g., a modification which enhances resistance to degradation,an iRNA agent having an architecture or structure described herein, aniRNA agent administered as described herein, or an iRNA agent formulatedas described herein, combined with, associated with, and delivered bysuch a drug delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features amodular complex. The complex can include a carrier agent linked to oneor more of (preferably two or more, more preferably all three of): (a) acondensing agent (e.g., an agent capable of attracting, e.g., binding, anucleic acid, e.g., through ionic or electrostatic interactions); (b) afusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane); and(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a cancer cell, endothelial cell or bonecell.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can belinked, e.g., coupled or bound, to the modular complex. The iRNA agentcan interact with the condensing agent of the complex, and the complexcan be used to deliver an iRNA agent to a cell, e.g., in vitro or invivo. For example, the complex can be used to deliver an iRNA agent to asubject in need thereof, e.g., to deliver an iRNA agent to a subjecthaving a disorder, e.g., a disorder described herein, such as a diseaseor disorder of the liver.

The fusogenic agent and the condensing agent can be different agents orthe one and the same agent. For example, a polyamino chain, e.g.,polyethyleneimine (PEI), can be the fusogenic and/or the condensingagent.

The delivery agent can be a modular complex. For example, the complexcan include a carrier agent linked to one or more of (preferably two ormore, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g.,binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or beingtransported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., alectin, glycoprotein, lipid or protein, e.g., an antibody, that binds toa specified cell type such as a cancer cell, endothelial cell, bonecell. A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGDpeptide or RGD peptide mimetic.

Carrier Agents

The carrier agent of a modular complex described herein can be asubstrate for attachment of one or more of: a condensing agent, afusogenic agent, and a targeting group. The carrier agent wouldpreferably lack an endogenous enzymatic activity. The agent wouldpreferably be a biological molecule, preferably a macromolecule.Polymeric biological carriers are preferred. It would also be preferredthat the carrier molecule be biodegradable.

The carrier agent can be a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier moleculecan also be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Other usefulcarrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least1 Da in size; (b) has at least 5 charged groups, preferably between 5and 5000 charged groups; (c) is present in the complex at a ratio of atleast 1:1 carrier agent to fusogenic agent; (d) is present in thecomplex at a ratio of at least 1:1 carrier agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 carrier agentto targeting agent.

Fusogenic Agents

A fusogenic agent of a modular complex described herein can be an agentthat is responsive to, e.g., changes charge depending on, the pHenvironment. Upon encountering the pH of an endosome, it can cause aphysical change, e.g., a change in osmotic properties which disrupts orincreases the permeability of the endosome membrane. Preferably, thefusogenic agent changes charge, e.g., becomes protonated, at pH lowerthan physiological range. For example, the fusogenic agent can becomeprotonated at pH 4.5-6.5. The fusogenic agent can serve to release theiRNA agent into the cytoplasm of a cell after the complex is taken up,e.g., via endocytosis, by the cell, thereby increasing the cellularconcentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an aminogroup, which, when exposed to a specified pH range, will undergo achange, e.g., in charge, e.g., protonation. The change in charge of thefusogenic agent can trigger a change, e.g., an osmotic change, in avesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, thefusogenic agent, upon being exposed to the pH environment of anendosome, will cause a solubility or osmotic change substantial enoughto increase the porosity of (preferably, to rupture) the endosomalmembrane.

The fusogenic agent can be a polymer, preferably a polyamino chain,e.g., polyethyleneimine (PEI). The PEI can be linear, branched,synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, orlipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine,polyimidazole, polypyridine, polypropyleneimine, mellitin, or apolyacetal substance, e.g., a cationic polyacetal. In some embodiment,the fusogenic agent can have an alpha helical structure. The fusogenicagent can be a membrane disruptive agent, e.g., mellittin.

A fusogenic agent can have one or more of the following characteristics:(a) is at least 1 Da in size; (b) has at least 10 charged groups,preferably between 10 and 5000 charged groups, more preferably between50 and 1000 charged groups; (c) is present in the complex at a ratio ofat least 1:1 fusogenic agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 fusogenic agent to condensing agent;(e) is present in the complex at a ratio of at least 1:1 fusogenic agentto targeting agent.

Other suitable fusogenic agents can be tested and identified by askilled artisan. The ability of a compound to respond to, e.g., changecharge depending on, the pH environment can be tested by routinemethods, e.g., in a cellular assay. For example, a test compound iscombined or contacted with a cell, and the cell is allowed to take upthe test compound, e.g., by endocytosis. An endosome preparation canthen be made from the contacted cells and the endosome preparationcompared to an endosome preparation from control cells. A change, e.g.,a decrease, in the endosome fraction from the contacted cell vs. thecontrol cell indicates that the test compound can function as afusogenic agent. Alternatively, the contacted cell and control cell canbe evaluated, e.g., by microscopy, e.g., by light or electronmicroscopy, to determine a difference in endosome population in thecells. The test compound can be labeled. In another type of assay, amodular complex described herein is constructed using one or more testor putative fusogenic agents. The modular complex can be constructedusing a labeled nucleic acid instead of the iRNA. The ability of thefusogenic agent to respond to, e.g., change charge depending on, the pHenvironment, once the modular complex is taken up by the cell, can beevaluated, e.g., by preparation of an endosome preparation, or bymicroscopy techniques, as described above. A two-step assay can also beperformed, wherein a first assay evaluates the ability of a testcompound alone to respond to, e.g., change charge depending on, the pHenvironment; and a second assay evaluates the ability of a modularcomplex that includes the test compound to respond to, e.g., changecharge depending on, the pH environment.

Condensing Agent

The condensing agent of a modular complex described herein can interactwith (e.g., attracts, holds, or binds to) an iRNA agent and act to (a)condense, e.g., reduce the size or charge of the iRNA agent and/or (b)protect the iRNA agent, e.g., protect the iRNA agent againstdegradation. The condensing agent can include a moiety, e.g., a chargedmoiety, that can interact with a nucleic acid, e.g., an iRNA agent,e.g., by ionic interactions. The condensing agent would preferably be acharged polymer, e.g., a polycationic chain. The condensing agent can bea polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least1 Da in size; (b) has at least 2 charged groups, preferably between 2and 100 charged groups; (c) is present in the complex at a ratio of atleast 1:1 condensing agent to carrier agent; (d) is present in thecomplex at a ratio of at least 1:1 condensing agent to fusogenic agent;(e) is present in the complex at a ratio of at least 1:1 condensingagent to targeting agent.

Other suitable condensing agents can be tested and identified by askilled artisan, e.g., by evaluating the ability of a test agent tointeract with a nucleic acid, e.g., an iRNA agent. The ability of a testagent to interact with a nucleic acid, e.g., an iRNA agent, e.g., tocondense or protect the iRNA agent, can be evaluated by routinetechniques. In one assay, a test agent is contacted with a nucleic acid,and the size and/or charge of the contacted nucleic acid is evaluated bya technique suitable to detect changes in molecular mass and/or charge.Such techniques include non-denaturing gel electrophoresis,immunological methods, e.g., immunoprecipitation, gel filtration, ionicinteraction chromatography, and the like. A test agent is identified asa condensing agent if it changes the mass and/or charge (preferablyboth) of the contacted nucleic acid, compared to a control. A two-stepassay can also be performed, wherein a first assay evaluates the abilityof a test compound alone to interact with, e.g., bind to, e.g., condensethe charge and/or mass of, a nucleic cid; and a second assay evaluatesthe ability of a modular complex that includes the test compound tointeract with, e.g., bind to, e.g., condense the charge and/or mass of,a nucleic acid.

Amphipathic Delivery Agents

In one aspect, the invention features an amphipathic delivery conjugateor module, such as those described herein and those described incopending, co-owned U.S. Provisional Application Ser. No. 60/455,050(Attorney Docket No. 14174-065P01), filed on Mar. 13, 2003, which ishereby incorporated by reference.

In addition, the invention include an iRNA agent described herein, e.g.,a palindromic iRNA agent, an iRNA agent having a non canonical pairing,an iRNA agent which targets a gene described herein, e.g., a gene activein the liver, an iRNA agent having a chemical modification describedherein, e.g., a modification which enhances resistance to degradation,an iRNA agent having an architecture or structure described herein, aniRNA agent administered as described herein, or an iRNA agent formulatedas described herein, combined with, associated with, and delivered bysuch an amphipathic delivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and ahydrophilic region. Such molecules can interact with (e.g., penetrate ordisrupt) lipids, e.g., a lipid bilayer of a cell. As such, they canserve as delivery agent for an associated (e.g., bound) iRNA (e.g., aniRNA or sRNA described herein). A preferred amphipathic molecule to beused in the compositions described herein (e.g., the amphipathic iRNAconstructs descriebd herein) is a polymer. The polymer may have asecondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide,e.g., a polypeptide having a secondary structure such that thepolypeptide has a hydrophilic and a hybrophobic face. The design ofamphipathic peptide structures (e.g., alpha-helical polypeptides) isroutine to one of skill in the art. For example, the followingreferences provide guidance: Grell et al. (2001) Protein design andfolding: template trapping of self-assembled helical bundles J Pept Sci7(3):146-51; Chen et al. (2002) Determination of stereochemistrystability coefficients of amino acid side-chains in an amphipathicalpha-helix J Pept Res 59(1):18-33; Iwata et al. (1994) Design andsynthesis of amphipathic 3(10)-helical peptides and their interactionswith phospholipid bilayers and ion channel formation J Biol Chem269(7):4928-33; Cornut et al. (1994) The amphipathic alpha-helixconcept. Application to the de novo design of ideally amphipathic Leu,Lys peptides with hemolytic activity higher than that of melittin FEBSLett 349(1):29-33; Negrete et al. (1998) Deciphering the structural codefor proteins: helical propensities in domain classes and statisticalmultiresidue information in alpha-helices. Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two ormore amphipathic subunits, e.g., two or more subunits containing cyclicmoieties (e.g., a cyclic moiety having one or more hydrophilic groupsand one or more hydrophobic groups). For example, the subunit maycontain a steroid, e.g., cholic acid; or a aromatic moiety. Suchmoieties preferably can exhibit atropisomerism, such that they can formopposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipidmembrane, e.g., a cell membrane, can be tested by routine methods, e.g.,in a cell free or cellular assay. For example, a test compound iscombined or contacted with a synthetic lipid bilayer, a cellularmembrane fraction, or a cell, and the test compound is evaluated for itsability to interact with, penetrate or disrupt the lipid bilayer, cellmembrane or cell. The test compound can labeled in order to detect theinteraction with the lipid bilayer, cell membrane or cell. In anothertype of assay, the test compound is linked to a reporter molecule or aniRNA agent (e.g., an iRNA or sRNA described herein) and the ability ofthe reporter molecule or iRNA agent to penetrate the lipid bilayer, cellmembrane or cell is evaluated. A two-step assay can also be performed,wherein a first assay evaluates the ability of a test compound alone tointeract with a lipid bilayer, cell membrane or cell; and a second assayevaluates the ability of a construct (e.g., a construct describedherein) that includes the test compound and a reporter or iRNA agent tointeract with a lipid bilayer, cell membrane or cell.

An amphipathic polymer useful in the compositions described herein hasat least 2, preferably at least 5, more preferably at least 10, 25, 50,100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids orcyclic subunits). A single amphipathic polymer can be linked to one ormore, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agentsdescribed herein). In some embodiments, an amphipathic polymer cancontain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g.,an iRNA or sRNA described herein) in association with an amphipathicmolecule. Such compositions may be referred to herein as “amphipathiciRNA constructs.” Such compositions and constructs are useful in thedelivery or targeting of iRNA agents, e.g., delivery or targeting ofiRNA agents to a cell. While not wanting to be bound by theory, suchcompositions and constructs can increase the porosity of, e.g., canpenetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g.,to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNAagent (e.g., an iRNA or sRNA agent described herein) linked to anamphipathic molecule. The iRNA agent and the amphipathic molecule may beheld in continuous contact with one another by either covalent ornoncovalent linkages.

The amphipathic molecule of the composition or construct is preferablyother than a phospholipid, e.g., other than a micelle, membrane ormembrane fragment.

The amphipathic molecule of the composition or construct is preferably apolymer. The polymer may include two or more amphipathic subunits. Oneor more hydrophilic groups and one or more hydrophobic groups may bepresent on the polymer. The polymer may have a repeating secondarystructure as well as a first face and a second face. The distribution ofthe hydrophilic groups and the hydrophobic groups along the repeatingsecondary structure can be such that one face of the polymer is ahydrophilic face and the other face of the polymer is a hydrophobicface.

The amphipathic molecule can be a polypeptide, e.g., a polypeptidecomprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunitscontaining one or more cyclic moiety (e.g., a cyclic moiety having oneor more hydrophilic groups and/or one or more hydrophobic groups). Inone embodiment, the polymer is a polymer of cyclic moieties such thatthe moieties have alternating hydrophobic and hydrophilic groups. Forexample, the subunit may contain a steroid, e.g., cholic acid. Inanother example, the subunit may contain an aromatic moiety. Thearomatic moiety may be one that can exhibit atropisomerism, e.g., a2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted)biphenyl. A subunit may include an aromatic moiety of Formula (M):

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted witharyl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted withO, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonicacid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo,nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonicacid, phosphate, phosphate ester, substituted or unsubstituted aryl,carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/oroptionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ froms a fused phenylring.

R₄ is hydrogen, or when taken together with R₃ froms a fused phenylring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl,alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl;or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionallysubstituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl oralkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester,substituted or unsubstituted aryl, carboxyl, carboxylate, aminocarbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S,S(O), SO₂, alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNAagent and a drug that affects the uptake of the iRNA agent into thecell. The drug can be administered before, after, or at the same timethat the iRNA agent is administered. The drug can be covalently linkedto the iRNA agent. The drug can be, for example, a lipopolysaccharide,an activator of p38 MAP kinase, or an activator of NF-κB. The drug canhave a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, forexample, by disrupting the cell's cytoskeleton, e.g., by disrupting thecell's microtubules, microfilaments, and/or intermediate filaments. Thedrug can be, for example, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary drug's thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a secondagent. For example, an iRNA agent used to treat a particular disordercan be coupled to a second therapeutic agent, e.g., an agent other thanthe iRNA agent. The second therapeutic agent can be one which isdirected to the treatment of the same disorder. For example, in the caseof an iRNA used to treat a disorder characterized by unwanted cellproliferation, e.g., cancer, the iRNA agent can be coupled to a secondagent which has an anti-cancer effect. For example, it can be coupled toan agent which stimulates the immune system, e.g., a CpG motif, or moregenerally an agent that activates a toll-like receptor and/or increasesthe production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods.Exemplary methods include: organic synthesis and RNA cleavage, e.g., invitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand ofa double-stranded RNA molecule. The component strands can then beannealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB(Uppsala Sweden), can be used to produce a large amount of a particularRNA strand for a given iRNA. The OligoPilotII reactor can efficientlycouple a nucleotide using only a 1.5 molar excess of a phosphoramiditenucleotide. To make an RNA strand, ribonucleotides amidites are used.Standard cycles of monomer addition can be used to synthesize the 21 to23 nucleotide strand for the iRNA. Typically, the two complementarystrands are produced separately and then annealed, e.g., after releasefrom the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. Thecomplementary of the species to a particular target gene can beprecisely specified. For example, the species may be complementary to aregion that includes a polymorphism, e.g., a single nucleotidepolymorphism. Further the location of the polymorphism can be preciselydefined. In some embodiments, the polymorphism is located in an internalregion, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of thetermini.

dsRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can bemediated in vitro or in vivo. For example, to produce iRNAs by cleavagein vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid(DNA) segment in both directions. For example, the HiScribe™ RNAitranscription kit (New England Biolabs) provides a vector and a methodfor producing a dsRNA for a nucleic acid segment that is cloned into thevector at a position flanked on either side by a T7 promoter. Separatetemplates are generated for T7 transcription of the two complementarystrands for the dsRNA. The templates are transcribed in vitro byaddition of T7 RNA polymerase and dsRNA is produced. Similar methodsusing PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) canalso be used. In one embodiment, RNA generated by this method iscarefully purified to remove endotoxins that may contaminatepreparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example,using a Dicer or comparable RNAse III-based activity. For example, thedsRNA can be incubated in an in vitro extract from Drosophila or usingpurified components, e.g. a purified RNAse or RISC complex (RNA-inducedsilencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15;15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, eachbeing a particular 21 to 23 nt fragment of a source dsRNA molecule. Forexample, iRNAs that include sequences complementary to overlappingregions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can beprepared in a solution (e.g., an aqueous and/or organic solution) thatis appropriate for formulation. For example, the iRNA preparation can beprecipitated and redissolved in pure double-distilled water, andlyophilized. The dried iRNA can then be resuspended in a solutionappropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussedbelow.

Formulation

The iRNA agents described herein can be formulated for administration toa subject

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In someexamples, the composition is at least partially crystalline, uniformlycrystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10%water). In another example, the iRNA is in an aqueous phase, e.g., in asolution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a microparticle as can beappropriate for a crystalline composition). Generally, the iRNAcomposition is formulated in a manner that is compatible with theintended method of administration (see, below).

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A iRNA preparation can be formulated in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a iRNA,e.g., a protein that complexes with iRNA to form an iRNP. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAseinhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent,e.g., a second iRNA that can mediated RNAi with respect to a secondgene, or with respect to the same gene. Still other preparation caninclude at least 3, 5, ten, twenty, fifty, or a hundred or moredifferent iRNA species. Such iRNAs can mediated RNAi with respect to asimilar number of different genes.

In one embodiment, the iRNA preparation includes at least a secondtherapeutic agent (e.g., an agent other than an RNA or a DNA). Forexample, a iRNA composition for the treatment of a viral disease, e.g.HIV, might include a known antiviral agent (e.g., a protease inhibitoror reverse transcriptase inhibitor). In another example, a iRNAcomposition for the treatment of a cancer might further comprise achemotherapeutic agent.

Exemplary formulations are discussed below:

Liposomes

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA s agents, and such practice is within the invention. AniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) preparation can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate RNAi. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in adetergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNApreparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA andcondense around the iRNA to form a liposome. After condensation, thedetergent is removed, e.g., by dialysis, to yield a liposomalpreparation of iRNA.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Further description of methods for producing stable polynucleotidedelivery vehicles, which incorporate a polynucleotide/cationic lipidcomplex as structural components of the delivery vehicle, are describedin, e.g., WO 96/37194. Liposome formation can also include one or moreaspects of exemplary methods described in Felgner, P. L. et al., Proc.Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355;5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al.Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci.75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA preparations intoliposomes.

Liposomes that are pH-sensitive or negatively-charged, entrap nucleicacid molecules rather than complex with them. Since both the nucleicacid molecules and the lipid are similarly charged, repulsion ratherthan complex formation occurs. Nevertheless, some nucleic acid moleculesare entrapped within the aqueous interior of these liposomes.pH-sensitive liposomes have been used to deliver DNA encoding thethymidine kinase gene to cell monolayers in culture. Expression of theexogenous gene was detected in the target cells (Zhou et al., Journal ofControlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNAs in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA (see, e.g., Feigner,P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S.Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA, into the skin. In some implementations,liposomes are used for delivering iRNA to epidermal cells and also toenhance the penetration of iRNA into dermal tissues, e.g., into skin.For example, the liposomes can be applied topically. Topical delivery ofdrugs formulated as liposomes to the skin has been documented (see,e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 anddu Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J.and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al.Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987;Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983;Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855,1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA can bedelivered, for example, subcutaneously by infection in order to deliveriRNA to keratinocytes in the skin. In order to cross intact mammalianskin, lipid vesicles must pass through a series of fine pores, each witha diameter less than 50 nm, under the influence of a suitabletransdermal gradient. In addition, due to the lipid properties, thesetransferosomes can be self-optimizing (adaptive to the shape of pores,e.g., in the skin), self-repairing, and can frequently reach theirtargets without fragmenting, and often self-loading. The iRNA agents caninclude an RRMS tethered to a moiety which improves association with aliposome.

Surfactants

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention.Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes (see above). iRNA (or aprecursor, e.g., a larger dsRNA which can be processed into a iRNA, or aDNA which encodes a iRNA or precursor) compositions can include asurfactant. In one embodiment, the iRNA is formulated as an emulsionthat includes a surfactant. The most common way of classifying andranking the properties of the many different types of surfactants, bothnatural and synthetic, is by the use of the hydrophile/lipophile balance(HLB). The nature of the hydrophilic group provides the most usefulmeans for categorizing the different surfactants used in formulations(Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., NewYork, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical products and are usable over a wide range of pH values.In general their HLB values range from 2 to about 18 depending on theirstructure. Nonionic surfactants include nonionic esters such as ethyleneglycol esters, propylene glycol esters, glyceryl esters, polyglycerylesters, sorbitan esters, sucrose esters, and ethoxylated esters.Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates,propoxylated alcohols, and ethoxylated/propoxylated block polymers arealso included in this class. The polyoxyethylene surfactants are themost popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and Other Membranous Formulations

For ease of exposition the micelles and other formulations, compositionsand methods in this section are discussed largely with regard tounmodified iRNA agents. It should be understood, however, that thesemicelles and other formulations, compositions and methods can bepracticed with other iRNA agents, e.g., modified iRNA agents, and suchpractice is within the invention. The iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof)) composition can be provided as a micellarformulation. “Micelles” are defined herein as a particular type ofmolecular assembly in which amphipathic molecules are arranged in aspherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the iRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing is preferred in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe iRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the iRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e. there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g. through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether.Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g. atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

The iRNA agents can include an RRMS tethered to a moiety which improvesassociation with a micelle or other membranous formulation.

Particles

For ease of exposition the particles, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these particles,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. In another embodiment, an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) preparations may be incorporated into a particle, e.g., amicroparticle. Microparticles can be produced by spray-drying, but mayalso be produced by other methods including lyophilization, evaporation,fluid bed drying, vacuum drying, or a combination of these techniques.See below for further description.

Sustained-Release Formulations. An iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) described herein can be formulated for controlled, e.g., slowrelease. Controlled release can be achieved by disposing the iRNA withina structure or substance which impedes its release. E.g., iRNA can bedisposed within a porous matrix or in an erodable matrix, either ofwhich allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as asustained-release reservoir of iRNA that is taken up by cells onlyreleased from the microparticle through biodegradation. The polymericparticles in this embodiment should therefore be large enough topreclude phagocytosis (e.g., larger than 10 μm and preferably largerthan 20 μm). Such particles can be produced by the same methods to makesmaller particles, but with less vigorous mixing of the first and secondemulsions. That is to say, a lower homogenization speed, vortex mixingspeed, or sonication setting can be used to obtain particles having adiameter around 100 μm rather than 10 The time of mixing also can bealtered.

Larger microparticles can be formulated as a suspension, a powder, or animplantable solid, to be delivered by intramuscular, subcutaneous,intradermal, intravenous, or intraperitoneal injection; via inhalation(intranasal or intrapulmonary); orally; or by implantation. Theseparticles are useful for delivery of any iRNA when slow release over arelatively long term is desired. The rate of degradation, andconsequently of release, varies with the polymeric formulation.

Microparticles preferably include pores, voids, hollows, defects orother interstitial spaces that allow the fluid suspension medium tofreely permeate or perfuse the particulate boundary. For example, theperforated microstructures can be used to form hollow, porous spraydried microspheres.

Polymeric particles containing iRNA (e.g., a sRNA) can be made using adouble emulsion technique, for instance. First, the polymer is dissolvedin an organic solvent. A preferred polymer is polylactic-co-glycolicacid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50,or 75:25. Next, a sample of nucleic acid suspended in aqueous solutionis added to the polymer solution and the two solutions are mixed to forma first emulsion. The solutions can be mixed by vortexing or shaking,and in a preferred method, the mixture can be sonicated. Most preferableis any method by which the nucleic acid receives the least amount ofdamage in the form of nicking, shearing, or degradation, while stillallowing the formation of an appropriate emulsion. For example,acceptable results can be obtained with a Vibra-cell model VC-250sonicator with a ⅛″ microtip probe, at setting #3.

Spray-Drying.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)) can beprepared by spray drying. Spray dried iRNA can be administered to asubject or be subjected to further formulation. A pharmaceuticalcomposition of iRNA can be prepared by spray drying a homogeneousaqueous mixture that includes a iRNA under conditions sufficient toprovide a dispersible powdered composition, e.g., a pharmaceuticalcomposition. The material for spray drying can also include one or moreof: a pharmaceutically acceptable excipient, or adispersibility-enhancing amount of a physiologically acceptable,water-soluble protein. The spray-dried product can be a dispersiblepowder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to adried particulate form. Spray drying can be used to provide powderedmaterial for various administrative routes including inhalation. See,for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols:Physical and Biological Basis for Therapy, A. J. Hickey, ed. MarcelDekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspensionto form a fine mist of droplets and drying the droplets. The mist can beprojected into a drying chamber (e.g., a vessel, tank, tubing, or coil)where it contacts a drying gas. The mist can include solid or liquidpore forming agents. The solvent and pore forming agents evaporate fromthe droplets into the drying gas to solidify the droplets,simultaneously forming pores throughout the solid. The solid (typicallyin a powder, particulate form) then is separated from the drying gas andcollected.

Spray drying includes bringing together a highly dispersed liquid, and asufficient volume of air (e.g., hot air) to produce evaporation anddrying of the liquid droplets. The preparation to be spray dried can beany solution, course suspension, slurry, colloidal dispersion, or pastethat may be atomized using the selected spray drying apparatus.Typically, the feed is sprayed into a current of warm filtered air thatevaporates the solvent and conveys the dried product to a collector. Thespent air is then exhausted with the solvent. Several different types ofapparatus may be used to provide the desired product. For example,commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. caneffectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape,nearly uniform in size and frequently hollow. There may be some degreeof irregularity in shape depending upon the incorporated medicament andthe spray drying conditions. In many instances the dispersion stabilityof spray-dried microspheres appears to be more effective if an inflatingagent (or blowing agent) is used in their production. Particularlypreferred embodiments may comprise an emulsion with an inflating agentas the disperse or continuous phase (the other phase being aqueous innature). An inflating agent is preferably dispersed with a surfactantsolution, using, for instance, a commercially available microfluidizerat a pressure of about 5000 to 15,000 psi. This process forms anemulsion, preferably stabilized by an incorporated surfactant, typicallycomprising submicron droplets of water immiscible blowing agentdispersed in an aqueous continuous phase. The formation of suchdispersions using this and other techniques are common and well known tothose in the art. The blowing agent is preferably a fluorinated compound(e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin,perfluorobutyl ethane) which vaporizes during the spray-drying process,leaving behind generally hollow, porous aerodynamically lightmicrospheres. As will be discussed in more detail below, other suitableblowing agents include chloroform, freons, and hydrocarbons. Nitrogengas and carbon dioxide are also contemplated as a suitable blowingagent.

Although the perforated microstructures are preferably formed using ablowing agent as described above, it will be appreciated that, in someinstances, no blowing agent is required and an aqueous dispersion of themedicament and surfactant(s) are spray dried directly. In such cases,the formulation may be amenable to process conditions (e.g., elevatedtemperatures) that generally lead to the formation of hollow, relativelyporous microparticles. Moreover, the medicament may possess specialphysicochemical properties (e.g., high crystallinity, elevated meltingtemperature, surface activity, etc.) that make it particularly suitablefor use in such techniques.

The perforated microstructures may optionally be associated with, orcomprise, one or more surfactants. Moreover, miscible surfactants mayoptionally be combined with the suspension medium liquid phase. It willbe appreciated by those skilled in the art that the use of surfactantsmay further increase dispersion stability, simplify formulationprocedures or increase bioavailability upon administration. Of coursecombinations of surfactants, including the use of one or more in theliquid phase and one or more associated with the perforatedmicrostructures are contemplated as being within the scope of theinvention. By “associated with or comprise” it is meant that thestructural matrix or perforated microstructure may incorporate, adsorb,absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition thataids in the formation and maintenance of the stabilized respiratorydispersions by forming a layer at the interface between the structuralmatrix and the suspension medium. The surfactant may comprise a singlecompound or any combination of compounds, such as in the case ofco-surfactants. Particularly preferred surfactants are substantiallyinsoluble in the propellant, nonfluorinated, and selected from the groupconsisting of saturated and unsaturated lipids, nonionic detergents,nonionic block copolymers, ionic surfactants, and combinations of suchagents. It should be emphasized that, in addition to the aforementionedsurfactants, suitable (i.e. biocompatible) fluorinated surfactants arecompatible with the teachings herein and may be used to provide thedesired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sourcesmay be used in varying concentrations to form a structural matrix.Generally, compatible lipids comprise those that have a gel to liquidcrystal phase transition greater than about 40° C. Preferably, theincorporated lipids are relatively long chain (i.e. C₆-C₂₂) saturatedlipids and more preferably comprise phospholipids. Exemplaryphospholipids useful in the disclosed stabilized preparations compriseegg phosphatidylcholine, dilauroylphosphatidylcholine,dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines,phosphatidylethanolamine, dioleylphosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol,glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid,cardiolipin; lipids bearing polymer chains such as, polyethylene glycol,chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearingsulfonated mono-, di-, and polysaccharides; fatty acids such as palmiticacid, stearic acid, and oleic acid; cholesterol, cholesterol esters, andcholesterol hemisuccinate. Due to their excellent biocompatibilitycharacteristics, phospholipids and combinations of phospholipids andpoloxamers are particularly suitable for use in the stabilizeddispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters includingsorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitanmonooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitanmonolaurate, and polyoxyethylene (20) sorbitan monooleate, oleylpolyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, laurylpolyoxyethylene (4) ether, glycerol esters, and sucrose esters. Othersuitable nonionic detergents can be easily identified using McCutcheon'sEmulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.).Preferred block copolymers include diblock and triblock copolymers ofpolyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic®F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionicsurfactants such as sodium sulfosuccinate, and fatty acid soaps may alsobe utilized. In preferred embodiments, the microstructures may compriseoleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants orlipids are preferred especially in the case of delivery of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). Examples of suitablecationic lipids include: DOTMA,N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationicamino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization,pressure atomization and two-fluid atomization can be used. Examples ofthe devices used in these processes include “Parubisu [phoneticrendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”,manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray DrierL-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,”manufactured by Okawara Kakoki Co., can be used for the method ofspraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry thesprayed material, it is recommended to use air, nitrogen gas or an inertgas. The temperature of the inlet of the gas used to dry the sprayedmaterials such that it does not cause heat deactivation of the sprayedmaterial. The range of temperatures may vary between about 50° C. toabout 200° C., preferably between about 50° C. and 100° C. Thetemperature of the outlet gas used to dry the sprayed material, may varybetween about 0° C. and about 150° C., preferably between 0° C. and 90°C., and even more preferably between 0° C. and 60° C.

The spray drying is done under conditions that result in substantiallyamorphous powder of homogeneous constitution having a particle size thatis respirable, a low moisture content and flow characteristics thatallow for ready aerosolization. Preferably the particle size of theresulting powder is such that more than about 98% of the mass is inparticles having a diameter of about 10 μm or less with about 90% of themass being in particles having a diameter less than 5 μm. Alternatively,about 95% of the mass will have particles with a diameter of less than10 μm with about 80% of the mass of the particles having a diameter ofless than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNApreparation may optionally be combined with pharmaceutical carriers orexcipients which are suitable for respiratory and pulmonaryadministration. Such carriers may serve simply as bulking agents when itis desired to reduce the iRNA concentration in the powder which is beingdelivered to a patient, but may also serve to enhance the stability ofthe iRNA compositions and to improve the dispersibility of the powderwithin a powder dispersion device in order to provide more efficient andreproducible delivery of the iRNA and to improve handlingcharacteristics of the iRNA such as flowability and consistency tofacilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spraydrying, i.e., by adding the carrier material to the purified bulksolution. In that way, the carrier particles will be formedsimultaneously with the drug particles to produce a homogeneous powder.Alternatively, the carriers may be separately prepared in a dry powderform and combined with the dry powder drug by blending. The powdercarriers will usually be crystalline (to avoid water absorption), butmight in some cases be amorphous or mixtures of crystalline andamorphous. The size of the carrier particles may be selected to improvethe flowability of the drug powder, typically being in the range from 25μm to 100 μm. A preferred carrier material is crystalline lactose havinga size in the above-stated range.

Powders prepared by any of the above methods will be collected from thespray dryer in a conventional manner for subsequent use. For use aspharmaceuticals and other purposes, it will frequently be desirable todisrupt any agglomerates which may have formed by screening or otherconventional techniques. For pharmaceutical uses, the dry powderformulations will usually be measured into a single dose, and the singledose sealed into a package. Such packages are particularly useful fordispersion in dry powder inhalers, as described in detail below.Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components aredescribed in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602;and 4,486,435. Bloch and Speison (1983) Pharm. Acta Helv 58:14-22teaches spray drying of hydrochlorothiazide and chlorthalidone(lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) inazeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A numberof Japanese Patent application Abstracts relate to spray drying ofhydrophilic-hydrophobic product combinations, including JP 806766; JP7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP4036233. Other foreign patent publications relevant to spray dryinghydrophilic-hydrophobic product combinations include FR 2594693; DE2209477; and WO 88/07870.

Lyophilization.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g.,a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof)preparation can be made by lyophilization. Lyophilization is afreeze-drying process in which water is sublimed from the compositionafter it is frozen. The particular advantage associated with thelyophilization process is that biologicals and pharmaceuticals that arerelatively unstable in an aqueous solution can be dried without elevatedtemperatures (thereby eliminating the adverse thermal effects), and thenstored in a dry state where there are few stability problems. Withrespect to the instant invention such techniques are particularlycompatible with the incorporation of nucleic acids in perforatedmicrostructures without compromising physiological activity. Methods forproviding lyophilized particulates are known to those of skill in theart and it would clearly not require undue experimentation to providedispersion compatible microstructures in accordance with the teachingsherein. Accordingly, to the extent that lyophilization processes may beused to provide microstructures having the desired porosity and size,they are conformance with the teachings herein and are expresslycontemplated as being within the scope of the instant invention.

Targeting

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAs. Itshould be understood, however, that these formulations, compositions andmethods can be practiced with other iRNA agents, e.g., modified iRNAagents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is targeted to a particular cell. For example, a liposome or particle orother structure that includes a iRNA can also include a targeting moietythat recognizes a specific molecule on a target cell. The targetingmoiety can be a molecule with a specific affinity for a target cell.Targeting moieties can include antibodies directed against a proteinfound on the surface of a target cell, or the ligand or areceptor-binding portion of a ligand for a molecule found on the surfaceof a target cell. For example, the targeting moiety can recognize acancer-specific antigen (e.g., CA15-3, CA19-9, CEA, or HER2/neu.) or aviral antigen, thus delivering the iRNA to a cancer cell or avirus-infected cell. Exemplary targeting moieties include antibodies(such as IgM, IgG, IgA, IgD, and the like, or a functional portionsthereof), ligands for cell surface receptors (e.g., ectodomainsthereof).

Table 3 provides a number of antigens which can be used to targetselected cells.

TABLE 3 ANTIGEN Exemplary tumor tissue CEA (carcinoembryonic antigen)colon, breast, lung PSA (prostate specific antigen) prostate cancerCA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancerHER2/neu breast cancer α-feto protein testicular cancer, hepatic cancerβ-HCG (human chorionic testicular cancer, choriocarcinoma gonadotropin)MUC-1 breast cancer Estrogen receptor breast cancer, uterine cancerProgesterone receptor breast cancer, uterine cancer EGFr (epidermalgrowth factor bladder cancer receptor)

In one embodiment, the targeting moiety is attached to a liposome. Forexample, U.S. Pat. No. 6,245,427 describes a method for targeting aliposome using a protein or peptide. In another example, a cationiclipid component of the liposome is derivatized with a targeting moiety.For example, WO 96/37194 describes convertingN-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimideactivated ester. The product was then coupled to an RGD peptide.

Genes and Diseases

In one aspect, the invention features, a method of treating a subject atrisk for or afflicted with unwanted cell proliferation, e.g., malignantor nonmalignant cell proliferation. The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein,e.g., an iRNA having a structure described herein, where the iRNA ishomologous to and can silence, e.g., by cleavage, a gene which promotesunwanted cell proliferation;

administering an iRNA agent, e.g., an sRNA or iRNA agent describedherein to a subject, preferably a human subject,

thereby treating the subject.

In a preferred embodiment the gene is a growth factor or growth factorreceptor gene, a kinase, e.g., a protein tyrosine, serine or threoninekinase gene, an adaptor protein gene, a gene encoding a G proteinsuperfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the iRNA agent silences the PDGF beta gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PDGF beta expression, e.g., testicular andlung cancers.

In another preferred embodiment the iRNA agent silences the Erb-B gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Erb-B expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the Src gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the iRNA agent silences the CRK gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted CRK expression, e.g., colon and lung cancers.

In a preferred embodiment the iRNA agent silences the GRB2 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted GRB2 expression, e.g., squamous cellcarcinoma.

In another preferred embodiment the iRNA agent silences the RAS gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted RAS expression, e.g., pancreatic, colon andlung cancers, and chronic leukemia.

In another preferred embodiment the iRNA agent silences the MEKK gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted MEKK expression, e.g., squamous cellcarcinoma, melanoma or leukemia.

In another preferred embodiment the iRNA agent silences the JNK gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted INK expression, e.g., pancreatic or breastcancers.

In a preferred embodiment the iRNA agent silences the RAF gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In a preferred embodiment the iRNA agent silences the Erk1/2 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the iRNA agent silences the PCNA(p21)gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted PCNA expression, e.g., lung cancer.

In a preferred embodiment the iRNA agent silences the MYB gene, and thuscan be used to treat a subject having or at risk for a disordercharacterized by unwanted MYB expression, e.g., colon cancer or chronicmyelogenous leukemia.

In a preferred embodiment the iRNA agent silences the c-MYC gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma orneuroblastoma.

In another preferred embodiment the iRNA agent silences the JUN gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted JUN expression, e.g., ovarian, prostate orbreast cancers.

In another preferred embodiment the iRNA agent silences the FOS gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted FOS expression, e.g., skin or prostatecancers.

In a preferred embodiment the iRNA agent silences the BCL-2 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted BCL-2 expression, e.g., lung or prostatecancers or Non-Hodgkin lymphoma.

In a preferred embodiment the iRNA agent silences the Cyclin D gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted Cyclin D expression, e.g., esophageal andcolon cancers.

In a preferred embodiment the iRNA agent silences the VEGF gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted VEGF expression, e.g., esophageal and coloncancers.

In a preferred embodiment the iRNA agent silences the EGFR gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the Cyclin Agene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin A expression, e.g., lung andcervical cancers.

In another preferred embodiment the iRNA agent silences the Cyclin Egene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Cyclin E expression, e.g., lung andbreast cancers.

In another preferred embodiment the iRNA agent silences the WNT-1 gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another preferred embodiment the iRNA agent silences the beta-cateningene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted beta-catenin expression, e.g.,adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the c-MET gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted c-MET expression, e.g., hepatocellularcarcinoma.

In another preferred embodiment the iRNA agent silences the PKC gene,and thus can be used to treat a subject having or at risk for a disordercharacterized by unwanted PKC expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the NFKB gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the STAT3 gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted STAT3 expression, e.g., prostate cancer.

In another preferred embodiment the iRNA agent silences the survivingene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted survivin expression, e.g., cervicalor pancreatic cancers.

In another preferred embodiment the iRNA agent silences the Her2/Neugene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Her2/Neu expression, e.g., breastcancer.

In another preferred embodiment the iRNA agent silences thetopoisomerase I gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted topoisomerase Iexpression, e.g., ovarian and colon cancers.

In a preferred embodiment the iRNA agent silences the topoisomerase IIalpha gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted topoisomerase II expression,e.g., breast and colon cancers.

In a preferred embodiment the iRNA agent silences mutations in the p73gene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted p73 expression, e.g., colorectaladenocarcinoma.

In a preferred embodiment the iRNA agent silences mutations in thep21(WAF1/CIP1) gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p21(WAF1/CIP1)expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in thep27(KIP1) gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted p27(KIP1) expression,e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the PPM1Dgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted PPM1D expression, e.g., breastcancer.

In a preferred embodiment the iRNA agent silences mutations in the RASgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted RAS expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences mutations in thecaveolin I gene, and thus can be used to treat a subject having or atrisk for a disorder characterized by unwanted caveolin I expression,e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences mutations in theMIB I gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted MIB I expression, e.g., malebreast carcinoma (MBC).

In another preferred embodiment the iRNA agent silences mutations in theMTAI gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted MTAI expression, e.g., ovariancarcinoma.

In another preferred embodiment the iRNA agent silences mutations in theM68 gene, and thus can be used to treat a subject having or at risk fora disorder characterized by unwanted M68 expression, e.g., humanadenocarcinomas of the esophagus, stomach, colon, and rectum.

In preferred embodiments the iRNA agent silences mutations in tumorsuppressor genes, and thus can be used as a method to promote apoptoticactivity in combination with chemotherapeutics.

In a preferred embodiment the iRNA agent silences mutations in the p53tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted p53 expression, e.g.,gall bladder, pancreatic and lung cancers.

In a preferred embodiment the iRNA agent silences mutations in the p53family member DN-p63, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted DN-p63 expression,e.g., squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the pRbtumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted pRb expression, e.g.,oral squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the APC1tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted APC1 expression, e.g.,colon cancer.

In a preferred embodiment the iRNA agent silences mutations in the BRCA1tumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted BRCA1 expression, e.g.,breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the PTENtumor suppressor gene, and thus can be used to treat a subject having orat risk for a disorder characterized by unwanted PTEN expression, e.g.,hamartomas, gliomas, and prostate and endometrial cancers.

In a preferred embodiment the iRNA agent silences MLL fusion genes,e.g., MLL-AF9, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted MLL fusion gene expression,e.g., acute leukemias.

In another preferred embodiment the iRNA agent silences the BCR/ABLfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted BCR/ABL fusion gene expression,e.g., acute and chronic leukemias.

In another preferred embodiment the iRNA agent silences the TEL/AML1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted TEL/AML1 fusion geneexpression, e.g., childhood acute leukemia.

In another preferred embodiment the iRNA agent silences the EWS/FLI1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted EWS/FLI1 fusion geneexpression, e.g., Ewing Sarcoma.

In another preferred embodiment the iRNA agent silences the TLS/FUS1fusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted TLS/FUS1 fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the PAX3/FKHRfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted PAX3/FKHR fusion geneexpression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the AML1/ETOfusion gene, and thus can be used to treat a subject having or at riskfor a disorder characterized by unwanted AML1/ETO fusion geneexpression, e.g., acute leukemia.

In another aspect, the invention features, a method of treating asubject, e.g., a human, at risk for or afflicted with a disease ordisorder that may benefit by angiogenesis inhibition e.g., cancer. Themethod includes:

providing an iRNA agent, e.g., an iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a gene which mediates angiogenesis;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the iRNA agent silences the alpha v-integringene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted alpha V integrin, e.g., brain tumorsor tumors of epithelial origin.

In a preferred embodiment the iRNA agent silences the Flt-1 receptorgene, and thus can be used to treat a subject having or at risk for adisorder characterized by unwanted Flt-1 receptors, eg. Cancer andrheumatoid arthritis.

In a preferred embodiment the iRNA agent silences the tubulin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. Cancer and retinalneovascularization.

In a preferred embodiment the iRNA agent silences the tubulin gene, andthus can be used to treat a subject having or at risk for a disordercharacterized by unwanted tubulin, eg. Cancer and retinalneovascularization.

In another aspect, the invention features a method of treating a subjectinfected with a virus or at risk for or afflicted with a disorder ordisease associated with a viral infection. The method includes:

providing an iRNA agent, e.g., and iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a viral gene of a cellular gene which mediates viralfunction, e.g., entry or growth;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

Thus, the invention provides for a method of treating patients infectedby the Human Papilloma Virus (HPV) or at risk for or afflicted with adisorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% ofcervical carcinomas and thus an antiviral therapy is an attractivemethod to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. Inanother preferred embodiment, the HPV gene is one of the group of E2,E6, or E7.

In a preferred embodiment the expression of a human gene that isrequired for HPV replication is reduced.

The invention also includes a method of treating patients infected bythe Human Immunodeficiency Virus (HIV) or at risk for or afflicted witha disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome(AIDS).

In a preferred embodiment, the expression of a HIV gene is reduced. Inanother preferred embodiment, the HIV gene is CCR5, Gag, or Rev.

In a preferred embodiment the expression of a human gene that isrequired for HIV replication is reduced. In another preferredembodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected bythe Hepatitis B Virus (HBV) or at risk for or afflicted with a disordermediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

In a preferred embodiment, the expression of a HBV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of the tail region of the HBV core protein, the pre-cregious(pre-c) region, or the cregious (c) region. In another preferredembodiment, a targeted HBV-RNA sequence is comprised of the poly(A)tail.

In preferred embodiment the expression of a human gene that is requiredfor HBV replication is reduced.

The invention also provides for a method of treating patients infectedby the Hepatitis A Virus (HAV), or at risk for or afflicted with adisorder mediated by HAV.

In a preferred embodiment the expression of a human gene that isrequired for HAV replication is reduced.

The present invention provides for a method of treating patientsinfected by the Hepatitis C Virus (HCV), or at risk for or afflictedwith a disorder mediated by HCV, e.g., cirrhosis

In a preferred embodiment, the expression of a HCV gene is reduced.

In another preferred embodiment the expression of a human gene that isrequired for HCV replication is reduced.

The present invention also provides for a method of treating patientsinfected by the any of the group of Hepatitis Viral strains comprisinghepatitis D, E, F, G, or H, or patients at risk for or afflicted with adisorder mediated by any of these strains of hepatitis.

In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, orH gene is reduced.

In another preferred embodiment the expression of a human gene that isrequired for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected bythe Respiratory Syncytial Virus (RSV) or at risk for or afflicted with adisorder mediated by RSV, e.g, lower respiratory tract infection ininfants and childhood asthma, pneumonia and other complications, e.g.,in the elderly.

In a preferred embodiment, the expression of a RSV gene is reduced. Inanother preferred embodiment, the targeted HBV gene encodes one of thegroup of genes N, L, or P.

In a preferred embodiment the expression of a human gene that isrequired for RSV replication is reduced.

Methods of the invention provide for treating patients infected by theHerpes Simplex Virus (HSV) or at risk for or afflicted with a disordermediated by HSV, e.g, genital herpes and cold sores as well aslife-threatening or sight-impairing disease mainly in immunocompromisedpatients.

In a preferred embodiment, the expression of a HSV gene is reduced. Inanother preferred embodiment, the targeted HSV gene encodes DNApolymerase or the helicase-primase.

In a preferred embodiment the expression of a human gene that isrequired for HSV replication is reduced.

The invention also provides a method for treating patients infected bythe herpes Cytomegalovirus (CMV) or at risk for or afflicted with adisorder mediated by CMV, e.g., congenital virus infections andmorbidity in immunocompromised patients.

In a preferred embodiment, the expression of a CMV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for CMV replication is reduced.

Methods of the invention also provide for a method of treating patientsinfected by the herpes Epstein Barr Virus (EBV) or at risk for orafflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma,non-Hodgkin lymphoma, and Hodgkin disease.

In a preferred embodiment, the expression of a EBV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for EBV replication is reduced.

Methods of the invention also provide for treating patients infected byKaposi's Sarcoma-associated Herpes Virus (KSHV), also called humanherpesvirus 8, or patients at risk for or afflicted with a disordermediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman'sdisease and AIDS-associated primary effusion lymphoma.

In a preferred embodiment, the expression of a KSHV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for KSHV replication is reduced.

The invention also includes a method for treating patients infected bythe JC Virus (JCV) or a disease or disorder associated with this virus,e.g., progressive multifocal leukoencephalopathy (PML).

In a preferred embodiment, the expression of a JCV gene is reduced.

In preferred embodiment the expression of a human gene that is requiredfor JCV replication is reduced.

Methods of the invention also provide for treating patients infected bythe myxovirus or at risk for or afflicted with a disorder mediated bymyxovirus, e.g., influenza.

In a preferred embodiment, the expression of a myxovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe rhinovirus or at risk for of afflicted with a disorder mediated byrhinovirus, e.g., the common cold.

In a preferred embodiment, the expression of a rhinovirus gene isreduced.

In preferred embodiment the expression of a human gene that is requiredfor rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe coronavirus or at risk for of afflicted with a disorder mediated bycoronavirus, e.g., the common cold.

In a preferred embodiment, the expression of a coronavirus gene isreduced.

In preferred embodiment the expression of a human gene that is requiredfor coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected bythe flavivirus West Nile or at risk for or afflicted with a disordermediated by West Nile Virus.

In a preferred embodiment, the expression of a West Nile Virus gene isreduced. In another preferred embodiment, the West Nile Virus gene isone of the group comprising E, NS3, or NS5.

In a preferred embodiment the expression of a human gene that isrequired for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe St. Louis Encephalitis flavivirus, or at risk for or afflicted witha disease or disorder associated with this virus, e.g., viralhaemorrhagic fever or neurological disease.

In a preferred embodiment, the expression of a St. Louis Encephalitisgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for St. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Tick-borne encephalitis flavivirus, or at risk for or afflicted witha disorder mediated by Tick-borne encephalitis virus, e.g., viralhaemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Tick-borne encephalitisvirus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Tick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patientsinfected by the Murray Valley encephalitis flavivirus, which commonlyresults in viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Murray Valleyencephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected bythe dengue flavivirus, or a disease or disorder associated with thisvirus, e.g., dengue haemorrhagic fever.

In a preferred embodiment, the expression of a dengue virus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for dengue virus replication is reduced.

Methods of the invention also provide for treating patients infected bythe Simian Virus 40 (SV40) or at risk for or afflicted with a disordermediated by SV40, e.g., tumorigenesis.

In a preferred embodiment, the expression of a SV40 gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for SV40 replication is reduced.

The invention also includes methods for treating patients infected bythe Human T Cell Lymphotropic Virus (HTLV), or a disease or disorderassociated with this virus, e.g., leukemia and myelopathy.

In a preferred embodiment, the expression of a HTLV gene is reduced. Inanother preferred embodiment the HTLV1 gene is the Tax transcriptionalactivator.

In a preferred embodiment the expression of a human gene that isrequired for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflictedwith a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected bythe encephalomyocarditis virus (EMCV) or at risk for or afflicted with adisorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditisin mice and pigs and is capable of infecting human myocardial cells.This virus is therefore a concern for patients undergoingxenotransplantation.

In a preferred embodiment, the expression of a EMCV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for EMCV replication is reduced.

The invention also includes a method for treating patients infected bythe measles virus (MV) or at risk for or afflicted with a disordermediated by MV, e.g. measles.

In a preferred embodiment, the expression of a MV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for MV replication is reduced.

The invention also includes a method for treating patients infected bythe Vericella zoster virus (VZV) or at risk for or afflicted with adisorder mediated by VZV, e.g. chicken pox or shingles (also calledzoster).

In a preferred embodiment, the expression of a VZV gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for VZV replication is reduced.

The invention also includes a method for treating patients infected byan adenovirus or at risk for or afflicted with a disorder mediated by anadenovirus, e.g. respiratory tract infection.

In a preferred embodiment, the expression of an adenovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for adenovirus replication is reduced.

The invention includes a method for treating patients infected by ayellow fever virus (YFV) or at risk for or afflicted with a disordermediated by a YFV, e.g. respiratory tract infection.

In a preferred embodiment, the expression of a YFV gene is reduced. Inanother preferred embodiment, the preferred gene is one of a group thatincludes the E, NS2A, or NS3 genes.

In a preferred embodiment the expression of a human gene that isrequired for YFV replication is reduced.

Methods of the invention also provide for treating patients infected bythe poliovirus or at risk for or afflicted with a disorder mediated bypoliovirus, e.g., polio.

In a preferred embodiment, the expression of a poliovirus gene isreduced.

In a preferred embodiment the expression of a human gene that isrequired for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected bya poxvirus or at risk for or afflicted with a disorder mediated by apoxvirus, e.g., smallpox In a preferred embodiment, the expression of apoxvirus gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subjectinfected with a pathogen, e.g., a bacterial, amoebic, parasitic, orfungal pathogen. The method includes:

providing a iRNA agent, e.g., a siRNA having a structure describedherein, where siRNA is homologous to and can silence, e.g., by cleavageof a pathogen gene;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

The target gene can be one involved in growth, cell wall synthesis,protein synthesis, transcription, energy metabolism, e.g., the Krebscycle, or toxin production.

Thus, the present invention provides for a method of treating patientsinfected by a plasmodium that causes malaria.

In a preferred embodiment, the expression of a plasmodium gene isreduced. In another preferred embodiment, the gene is apical membraneantigen 1 (AMA1).

In a preferred embodiment the expression of a human gene that isrequired for plasmodium replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium ulcerans, or a disease or disorder associated withthis pathogen, e.g. Buruli ulcers.

In a preferred embodiment, the expression of a Mycobacterium ulceransgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium tuberculosis, or a disease or disorder associated withthis pathogen, e.g. tuberculosis.

In a preferred embodiment, the expression of a Mycobacteriumtuberculosis gene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected bythe Mycobacterium leprae, or a disease or disorder associated with thispathogen, e.g. leprosy.

In a preferred embodiment, the expression of a Mycobacterium leprae geneis reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycobacterium leprae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Staphylococcus aureus, or a disease or disorder associatedwith this pathogen, e.g. infections of the skin and muscous membranes.

In a preferred embodiment, the expression of a Staphylococcus aureusgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pneumoniae, or a disease or disorderassociated with this pathogen, e.g. pneumonia or childhood lowerrespiratory tract infection.

In a preferred embodiment, the expression of a Streptococcus pneumoniaegene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Streptococcus pyogenes, or a disease or disorder associatedwith this pathogen, e.g. Strep throat or Scarlet fever.

In a preferred embodiment, the expression of a Streptococcus pyogenesgene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Chlamydia pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection

In a preferred embodiment, the expression of a Chlamydia pneumoniae geneis reduced.

In a preferred embodiment the expression of a human gene that isrequired for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected bythe bacteria Mycoplasma pneumoniae, or a disease or disorder associatedwith this pathogen, e.g. pneumonia or childhood lower respiratory tractinfection

In a preferred embodiment, the expression of a Mycoplasma pneumoniaegene is reduced.

In a preferred embodiment the expression of a human gene that isrequired for Mycoplasma pneumoniae replication is reduced.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a disease or disordercharacterized by an unwanted immune response, e.g., an inflammatorydisease or disorder, or an autoimmune disease or disorder. The methodincludes:

providing an iRNA agent, e.g., an iRNA agent having a structuredescribed herein, which iRNA agent is homologous to and can silence,e.g., by cleavage, a gene which mediates an unwanted immune response;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is an ischemia orreperfusion injury, e.g., ischemia or reperfusion injury associated withacute myocardial infarction, unstable angina, cardiopulmonary bypass,surgical intervention e.g., angioplasty, e.g., percutaneous transluminalcoronary angioplasty, the response to a transplanted organ or tissue,e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In a preferred embodiment the disease or disorder is restenosis, e.g.,restenosis associated with surgical intervention e.g., angioplasty,e.g., percutaneous transluminal coronary angioplasty.

In a preferred embodiment the disease or disorder is Inflammatory BowelDisease, e.g., Crohn Disease or Ulcerative Colitis.

In a preferred embodiment the disease or disorder is inflammationassociated with an infection or injury.

In a preferred embodiment the disease or disorder is asthma, lupus,multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g.,rheumatoid or psoriatic.

In particularly preferred embodiments the iRNA agent silences anintegrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

In particularly preferred embodiments the iRNA agent silences a selectinor co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin,P-selectin glycoprotein-1 (PSGL-1).

In particularly preferred embodiments the iRNA agent silences acomponent of the complement system, e.g., C3, C5, C3aR, C5aR, C3convertase, C5 convertase.

In particularly preferred embodiments the iRNA agent silences achemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2,IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3,PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES,MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, 1-309.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with acute pain or chronic pain.The method includes:

providing an iRNA agent, which iRNA is homologous to and can silence,e.g., by cleavage, a gene which mediates the processing of pain;

administering the iRNA to a subject,

thereby treating the subject.

In particularly preferred embodiments the iRNA agent silences acomponent of an ion channel.

In particularly preferred embodiments the iRNA agent silences aneurotransmitter receptor or ligand.

In one aspect, the invention features, a method of treating a subject,e.g., a human, at risk for or afflicted with a neurological disease ordisorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence,e.g., by cleavage, a gene which mediates a neurological disease ordisorder;

administering the to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is Alzheimer Diseaseor Parkinson Disease.

In particularly preferred embodiments the iRNA agent silences anamyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 andPSEN2, or I-synuclein.

In a preferred embodiment the disease or disorder is a neurodegenerativetrinucleotide repeat disorder, e.g., Huntington disease, dentatorubralpallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2,SCA3 (Machado-Joseph disease), SCA7 or SCA8.

In particularly preferred embodiments the iRNA agent silences HD, DRPLA,SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.

The loss of heterozygosity (LOH) can result in hemizygosity forsequence, e.g., genes, in the area of LOH. This can result in asignificant genetic difference between normal and disease-state cells,e.g., cancer cells, and provides a useful difference between normal anddisease-state cells, e.g., cancer cells. This difference can arisebecause a gene or other sequence is heterozygous in euploid cells but ishemizygous in cells having LOH. The regions of LOH will often include agene, the loss of which promotes unwanted proliferation, e.g., a tumorsuppressor gene, and other sequences including, e.g., other genes, insome cases a gene which is essential for normal function, e.g., growth.Methods of the invention rely, in part, on the specific cleavage orsilencing of one allele of an essential gene with an iRNA agent of theinvention. The iRNA agent is selected such that it targets the singleallele of the essential gene found in the cells having LOH but does notsilence the other allele, which is present in cells which do not showLOH. In essence, it discriminates between the two alleles,preferentially silencing the selected allele. In essence polymorphisms,e.g., SNPs of essential genes that are affected by LOH, are used as atarget for a disorder characterized by cells having LOH, e.g., cancercells having LOH.

E.g., one of ordinary skill in the art can identify essential geneswhich are in proximity to tumor suppressor genes, and which are within aLOH region which includes the tumor suppressor gene. The gene encodingthe large subunit of human RNA polymerase II, POLR2A, a gene located inclose proximity to the tumor suppressor gene p53, is such a gene. Itfrequently occurs within a region of LOH in cancer cells. Other genesthat occur within LOH regions and are lost in many cancer cell typesinclude the group comprising replication protein A 70-kDa subunit,replication protein A 32-kD, ribonucleotide reductase, thymidilatesynthase, TATA associated factor 2H, ribosomal protein S14, eukaryoticinitiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase,NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, the invention features, a method of treating a disordercharacterized by LOH, e.g., cancer. The method includes:

optionally, determining the genotype of the allele of a gene in theregion of LOH and preferably determining the genotype of both alleles ofthe gene in a normal cell;

providing an iRNA agent which preferentially cleaves or silences theallele found in the LOH cells;

administering the iRNA to the subject,

thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNAagent which can preferentially silence, e.g., cleave, one allele of apolymorphic gene

In another aspect, the invention provides a method of cleaving orsilencing more than one gene with an iRNA agent. In these embodimentsthe iRNA agent is selected so that it has sufficient homology to asequence found in more than one gene. For example, the sequenceAAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:6736) is conserved between mouselamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNAagent targeted to this sequence would effectively silence the entirecollection of genes.

The invention also includes an iRNA agent disclosed herein, which cansilence more than one gene.

Route of Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes a iRNA can be delivered to a subject by avariety of routes. Exemplary routes include: intravenous, topical,rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of iRNA and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the iRNA in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

For ocular administration, ointments or droppable liquids may bedelivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. In apreferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent,or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which canbe processed into a sRNA agent, or a DNA which encodes an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)is delivered to a subject via topical administration. “Topicaladministration” refers to the delivery to a subject by contacting theformulation directly to a surface of the subject. The most common formof topical delivery is to the skin, but a composition disclosed hereincan also be directly applied to other surfaces of the body, e.g., to theeye, a mucous membrane, to surfaces of a body cavity or to an internalsurface. As mentioned above, the most common topical delivery is to theskin. The term encompasses several routes of administration including,but not limited to, topical and transdermal. These modes ofadministration typically include penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

The term “skin,” as used herein, refers to the epidermis and/or dermisof an animal. Mammalian skin consists of two major, distinct layers. Theouter layer of the skin is called the epidermis. The epidermis iscomprised of the stratum corneum, the stratum granulosum, the stratumspinosum, and the stratum basale, with the stratum corneum being at thesurface of the skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm and 0.2 mm thick,depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker thanthe epidermis. The dermis is primarily composed of collagen in the formof fibrous bundles. The collagenous bundles provide support for, interalia, blood vessels, lymph capillaries, glands, nerve endings andimmunologically active cells.

One of the major functions of the skin as an organ is to regulate theentry of substances into the body. The principal permeability barrier ofthe skin is provided by the stratum corneum, which is formed from manylayers of cells in various states of differentiation. The spaces betweencells in the stratum corneum is filled with different lipids arranged inlattice-like formations that provide seals to further enhance the skinspermeability barrier.

The permeability barrier provided by the skin is such that it is largelyimpermeable to molecules having molecular weight greater than about 750Da. For larger molecules to cross the skin's permeability barrier,mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administeredagents. These factors include the characteristics of the treated skin,the characteristics of the delivery agent, interactions between both thedrug and delivery agent and the drug and skin, the dosage of the drugapplied, the form of treatment, and the post treatment regimen. Toselectively target the epidermis and dermis, it is sometimes possible toformulate a composition that comprises one or more penetration enhancersthat will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biologicalmembranes under the influence of an electric field) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163),phonophoresis or sonophoresis (use of ultrasound to enhance theabsorption of various therapeutic agents across biological membranes,notably the skin and the cornea) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 166), and optimization ofvehicle characteristics relative to dose position and retention at thesite of administration (Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 168) may be useful methods for enhancing thetransport of topically applied compositions across skin and mucosalsites.

The compositions and methods provided may also be used to examine thefunction of various proteins and genes in vitro in cultured or preserveddermal tissues and in animals. The invention can be thus applied toexamine the function of any gene. The methods of the invention can alsobe used therapeutically or prophylactically. For example, for thetreatment of animals that are known or suspected to suffer from diseasessuch as psoriasis, lichen planus, toxic epidermal necrolysis, ertythemamultiforme, basal cell carcinoma, squamous cell carcinoma, malignantmelanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lymedisease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Acomposition that includes an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) can be administered to a subject by pulmonary delivery.Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably iRNA, withinthe dispersion can reach the lung where it can be readily absorbedthrough the alveolar region directly into blood circulation. Pulmonarydelivery can be effective both for systemic delivery and for localizeddelivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self contained. Dry powder dispersion devices, for example,deliver drugs that may be readily formulated as dry powders. A iRNAcomposition may be stably stored as lyophilized or spray-dried powdersby itself or in combination with suitable powder carriers. The deliveryof a composition for inhalation can be mediated by a dosing timingelement which can include a timer, a dose counter, time measuringdevice, or a time indicator which when incorporated into the deviceenables dose tracking, compliance monitoring, and/or dose triggering toa patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 Usually the particle size distribution is between about 0.1 μmand about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The term “therapeutically effective amount” is the amount present in thecomposition that is needed to provide the desired level of drug in thesubject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered toa subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carriercan be taken into the lungs with no significant adverse toxicologicaleffects on the lungs.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatiblecarbohydrates, polypeptides, amino acids or combinations thereof.Suitable carbohydrates include monosaccharides such as galactose,D-mannose, sorbose, and the like; disaccharides, such as lactose,trehalose, and the like; cyclodextrins, such as2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such asraffinose, maltodextrins, dextrans, and the like; alditols, such asmannitol, xylitol, and the like. A preferred group of carbohydratesincludes lactose, threhalose, raffinose maltodextrins, and mannitol.Suitable polypeptides include aspartame. Amino acids include alanine andglycine, with glycine being preferred.

Additives, which are minor components of the composition of thisinvention, may be included for conformational stability during spraydrying and for improving dispersibility of the powder. These additivesinclude hydrophobic amino acids such as tryptophan, tyrosine, leucine,phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to unmodified iRNAagents. It should be understood, however, that these formulations,compositions and methods can be practiced with other iRNA agents, e.g.,modified iRNA agents, and such practice is within the invention. Boththe oral and nasal membranes offer advantages over other routes ofadministration. For example, drugs administered through these membraneshave a rapid onset of action, provide therapeutic plasma levels, avoidfirst pass effect of hepatic metabolism, and avoid exposure of the drugto the hostile gastrointestinal (GI) environment. Additional advantagesinclude easy access to the membrane sites so that the drug can beapplied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many drugs. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears tobe related to molecular size, lipid solubility and peptide proteinionization. Small molecules, less than 1000 daltons appear to crossmucosa rapidly. As molecular size increases, the permeability decreasesrapidly. Lipid soluble compounds are more permeable than non-lipidsoluble molecules. Maximum absorption occurs when molecules areun-ionized or neutral in electrical charges. Therefore charged moleculespresent the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to thebuccal cavity of a human being by spraying into the cavity, withoutinhalation, from a metered dose spray dispenser, a mixed micellarpharmaceutical formulation as described above and a propellant. In oneembodiment, the dispenser is first shaken prior to spraying thepharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions andmethods in this section are discussed largely with regard to unmodifiediRNA agents. It should be understood, however, that these devices,formulations, compositions and methods can be practiced with other iRNAagents, e.g., modified iRNA agents, and such practice is within theinvention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) canbe disposed on or in a device, e.g., a device which implanted orotherwise placed in a subject. Exemplary devices include devices whichare introduced into the vasculature, e.g., devices inserted into thelumen of a vascular tissue, or which devices themselves form a part ofthe vasculature, including stents, catheters, heart valves, and othervascular devices. These devices, e.g., catheters or stents, can beplaced in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to aiRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and otherorthopedic implants.

In one embodiment, unit doses or measured doses of a composition thatincludes iRNA are dispensed by an implanted device. The device caninclude a sensor that monitors a parameter within a subject. Forexample, the device can include pump, e.g., and, optionally, associatedelectronics.

Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) ex vivo and then administered or implantedin a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g.,tissue can be treated to reduce graft v. host disease. In otherembodiments, the tissue is allogeneic and the tissue is treated to treata disorder characterized by unwanted gene expression in that tissue.E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoieticcells, can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies.

In some implementations, the iRNA treated cells are insulated from othercells, e.g., by a semi-permeable porous barrier that prevents the cellsfrom leaving the implant, but enables molecules from the body to reachthe cells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains aniRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). Exemplary devicesinclude condoms, diaphragms, IUD (implantable uterine devices, sponges,vaginal sheaths, and birth control devices. In one embodiment, the iRNAis chosen to inactive sperm or egg. In another embodiment, the iRNA ischosen to be complementary to a viral or pathogen RNA, e.g., an RNA ofan STD. In some instances, the iRNA composition can include aspermicide.

Dosage

In one aspect, the invention features a method of administering an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject(e.g., a human subject). The method includes administering a unit doseof the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agentthat (a) the double-stranded part is 19-25 nucleotides (nt) long,preferably 21-23 nt, (b) is complementary to a target RNA (e.g., anendogenous or pathogen target RNA), and, optionally, (c) includes atleast one 3′ overhang 1-5 nucleotide long. In one embodiment, the unitdose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1,0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g.about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750,300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015,0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with thetarget RNA. The unit dose, for example, can be administered by injection(e.g., intravenous or intramuscular), an inhaled dose, or a topicalapplication. Particularly preferred dosages are less than 2, 1, or 0.1mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequentlythan once a day, e.g., less than every 2, 4, 8 or 30 days. In anotherembodiment, the unit dose is not administered with a frequency (e.g.,not a regular frequency). For example, the unit dose may be administereda single time.

In one embodiment, the effective dose is administered with othertraditional therapeutic modalities. In one embodiment, the subject has aviral infection and the modality is an antiviral agent other than aniRNA agent, e.g., other than a double-stranded iRNA agent, or sRNAagent. In another embodiment, the subject has atherosclerosis and theeffective dose of an iRNA agent, e.g., a double-stranded iRNA agent, orsRNA agent, is administered in combination with, e.g., after surgicalintervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an iRNA agent, e.g., a double-stranded iRNAagent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof). The maintenance dose or doses are generally lower than theinitial dose, e.g., one-half less of the initial dose. A maintenanceregimen can include treating the subject with a dose or doses rangingfrom 0.01 μs to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1,0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenancedoses are preferably administered no more than once every 5, 10, or 30days. Further, the treatment regimen may last for a period of time whichwill vary depending upon the nature of the particular disease, itsseverity and the overall condition of the patient. In preferredembodiments the dosage may be delivered no more than once per day, e.g.,no more than once per 24, 36, 48, or more hours, e.g., no more than oncefor every 5 or 8 days. Following treatment, the patient can be monitoredfor changes in his condition and for alleviation of the symptoms of thedisease state. The dosage of the compound may either be increased in theevent the patient does not respond significantly to current dosagelevels, or the dose may be decreased if an alleviation of the symptomsof the disease state is observed, if the disease state has been ablated,or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes aplurality of iRNA agent species. In another embodiment, the iRNA agentspecies has sequences that are non-overlapping and non-adjacent toanother species with respect to a naturally occurring target sequence.In another embodiment, the plurality of iRNA agent species is specificfor different naturally occurring target genes. In another embodiment,the iRNA agent is allele specific.

In some cases, a patient is treated with a iRNA agent in conjunctionwith other therapeutic modalities. For example, a patient being treatedfor a viral disease, e.g. an HIV associated disease (e.g., AIDS), may beadministered a iRNA agent specific for a target gene essential to thevirus in conjunction with a known antiviral agent (e.g., a proteaseinhibitor or reverse transcriptase inhibitor). In another example, apatient being treated for cancer may be administered a iRNA agentspecific for a target essential for tumor cell proliferation inconjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight(see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficientto be effective in treating or preventing a disorder or to regulate aphysiological condition in humans. The concentration or amount of iRNAagent administered will depend on the parameters determined for theagent and the method of administration, e.g. nasal, buccal, pulmonary.For example, nasal formulations tend to require much lowerconcentrations of some ingredients in order to avoid irritation orburning of the nasal passages. It is sometimes desirable to dilute anoral formulation up to 10-100 times in order to provide a suitable nasalformulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) can include a single treatment or,preferably, can include a series of treatments. It will also beappreciated that the effective dosage of a iRNA agent such as a sRNAagent used for treatment may increase or decrease over the course of aparticular treatment. Changes in dosage may result and become apparentfrom the results of diagnostic assays as described herein. For example,the subject can be monitored after administering a iRNA agentcomposition. Based on information from the monitoring, an additionalamount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human gene, e.g. a gene thatproduces a target RNA. The transgenic animal can be deficient for thecorresponding endogenous RNA. In another embodiment, the composition fortesting includes a iRNA agent that is complementary, at least in aninternal region, to a sequence that is conserved between the target RNAin the animal model and the target RNA in a human.

The inventors have discovered that iRNA agents described herein can beadministered to mammals, particularly large mammals such as nonhumanprimates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, composition is parenteral,e.g. intravenous (e.g., as a bolus or as a diffusible infusion),intradermal, intraperitoneal, intramuscular, intrathecal,intraventricular, intracranial, subcutaneous, transmucosal, buccal,sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary,intranasal, urethral or ocular. Administration can be provided by thesubject or by another person, e.g., a health care provider. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose. Selected modes of delivery are discussed inmore detail below.

The invention provides methods, compositions, and kits, for rectaladministration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes a an iRNA agent,e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)described herein, e.g., a therapeutically effective amount of a iRNAagent described herein, e.g., a iRNA agent having a double strandedregion of less than 40, and preferably less than 30 nucleotides andhaving one or two 1-3 nucleotide single strand 3′ overhangs can beadministered rectally, e.g., introduced through the rectum into thelower or upper colon. This approach is particularly useful in thetreatment of, inflammatory disorders, disorders characterized byunwanted cell proliferation, e.g., polyps, or colon cancer.

The medication can be delivered to a site in the colon by introducing adispensing device, e.g., a flexible, camera-guided device similar tothat used for inspection of the colon or removal of polyps, whichincludes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. TheiRNA agent of the enema can be dissolved in a saline or bufferedsolution. The rectal administration can also by means of a suppository,which can include other ingredients, e.g., an excipient, e.g., cocoabutter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally,e.g., in the form of tablets, capsules, gel capsules, lozenges, trochesor liquid syrups. Further, the composition can be applied topically to asurface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally.For example, the medication can be sprayed into the buccal cavity orapplied directly, e.g., in a liquid, solid, or gel form to a surface inthe buccal cavity. This administration is particularly desirable for thetreatment of inflammations of the buccal cavity, e.g., the gums ortongue, e.g., in one embodiment, the buccal administration is byspraying into the cavity, e.g., without inhalation, from a dispenser,e.g., a metered dose spray dispenser that dispenses the pharmaceuticalcomposition and a propellant.

Any of the iRNA agents described herein can be administered to oculartissue. For example, the medications can be applied to the surface ofthe eye or nearby tissue, e.g., the inside of the eyelid. They can beapplied topically, e.g., by spraying, in drops, as an eyewash, or anointment. Administration can be provided by the subject or by anotherperson, e.g., a health care provider. The medication can be provided inmeasured doses or in a dispenser which delivers a metered dose. Themedication can also be administered to the interior of the eye, and canbe introduced by a needle or other delivery device which can introduceit to a selected area or structure. Ocular treatment is particularlydesirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly tothe skin. For example, the medication can be applied topically ordelivered in a layer of the skin, e.g., by the use of a microneedle or abattery of microneedles which penetrate into the skin, but preferablynot into the underlying muscle tissue. Administration of the iRNA agentcomposition can be topical. Topical applications can, for example,deliver the composition to the dermis or epidermis of a subject. Topicaladministration can be in the form of transdermal patches, ointments,lotions, creams, gels, drops, suppositories, sprays, liquids or powders.A composition for topical administration can be formulated as aliposome, micelle, emulsion, or other lipophilic molecular assembly. Thetransdermal administration can be applied with at least one penetrationenhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to thepulmonary system. Pulmonary administration can be achieved by inhalationor by the introduction of a delivery device into the pulmonary system,e.g., by introducing a delivery device which can dispense themedication. A preferred method of pulmonary delivery is by inhalation.The medication can be provided in a dispenser which delivers themedication, e.g., wet or dry, in a form sufficiently small such that itcan be inhaled. The device can deliver a metered dose of medication. Thesubject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directlyaffect pulmonary tissue, but also for disorders which affect othertissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally.Nasal administration can be achieved by introduction of a deliverydevice into the nose, e.g., by introducing a delivery device which candispense the medication. Methods of nasal delivery include spray,aerosol, liquid, e.g., by drops, or by topical administration to asurface of the nasal cavity. The medication can be provided in adispenser with delivery of the medication, e.g., wet or dry, in a formsufficiently small such that it can be inhaled. The device can deliver ametered dose of medication. The subject, or another person, canadminister the medication.

Nasal delivery is effective not only for disorders which directly affectnasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder,crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in achemically or enzymatically produced artificial capsid or structurederived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can beadministered in order to alleviate the symptoms of a disease state,e.g., cancer or a cardiovascular disease. A subject can be treated withthe pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering apharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent) composition that is in a powdered form, e.g., a collection ofmicroparticles, such as crystalline particles. The composition caninclude a plurality of iRNA agents, e.g., specific for one or moredifferent endogenous target RNAs. The method can include other featuresdescribed herein.

A subject can be treated by administering a defined amount of an iRNAagent composition that is prepared by a method that includesspray-drying, i.e. atomizing a liquid solution, emulsion, or suspension,immediately exposing the droplets to a drying gas, and collecting theresulting porous powder particles. The composition can include aplurality of iRNA agents, e.g., specific for one or more differentendogenous target RNAs. The method can include other features describedherein.

The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent,(e.g., a precursor, e.g., a larger iRNA agent which can be processedinto a sRNA agent, or a DNA which encodes an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, or precursor thereof), can beprovided in a powdered, crystallized or other finely divided form, withor without a carrier, e.g., a micro- or nano-particle suitable forinhalation or other pulmonary delivery. This can include providing anaerosol preparation, e.g., an aerosolized spray-dried composition. Theaerosol composition can be provided in and/or dispensed by a metereddose delivery device.

The subject can be treated for a condition treatable by inhalation,e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agentwhich can be processed into a sRNA agent, or a DNA which encodes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursorthereof) composition and inhaling the aerosolized composition. The iRNAagent can be an sRNA. The composition can include a plurality of iRNAagents, e.g., specific for one or more different endogenous target RNAs.The method can include other features described herein.

A subject can be treated by, for example, administering a compositionincluding an effective/defined amount of an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof), wherein the composition is prepared by amethod that includes spray-drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes:evaluating a parameter related to the abundance of a transcript in acell of a subject; comparing the evaluated parameter to a referencevalue; and if the evaluated parameter has a preselected relationship tothe reference value (e.g., it is greater), administering a iRNA agent(or a precursor, e.g., a larger iRNA agent which can be processed into asRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) tothe subject. In one embodiment, the iRNA agent includes a sequence thatis complementary to the evaluated transcript. For example, the parametercan be a direct measure of transcript levels, a measure of a proteinlevel, a disease or disorder symptom or characterization (e.g., rate ofcell proliferation and/or tumor mass, viral load)

In another aspect, the invention features a method that includes:administering a first amount of a composition that comprises an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) to a subject, whereinthe iRNA agent includes a strand substantially complementary to a targetnucleic acid; evaluating an activity associated with a protein encodedby the target nucleic acid; wherein the evaluation is used to determineif a second amount should be administered. In a preferred embodiment themethod includes administering a second amount of the composition,wherein the timing of administration or dosage of the second amount is afunction of the evaluating. The method can include other featuresdescribed herein.

In another aspect, the invention features a method of administering asource of a double-stranded iRNA agent (ds iRNA agent) to a subject. Themethod includes administering or implanting a source of a ds iRNA agent,e.g., a sRNA agent, that (a) includes a double-stranded region that is19-25 nucleotides long, preferably 21-23 nucleotides, (b) iscomplementary to a target RNA (e.g., an endogenous RNA or a pathogenRNA), and, optionally, (c) includes at least one 3′ overhang 1-5 ntlong. In one embodiment, the source releases ds iRNA agent over time,e.g. the source is a controlled or a slow release source, e.g., amicroparticle that gradually releases the ds iRNA agent. In anotherembodiment, the source is a pump, e.g., a pump that includes a sensor ora pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof)including a nucleotide sequence complementary to a target RNA, e.g.,substantially and/or exactly complementary. The target RNA can be atranscript of an endogenous human gene. In one embodiment, the iRNAagent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b)is complementary to an endogenous target RNA, and, optionally, (c)includes at least one 3′ overhang 1-5 nt long. In one embodiment, thepharmaceutical composition can be an emulsion, microemulsion, cream,jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agentmixed with a topical delivery agent. The topical delivery agent can be aplurality of microscopic vesicles. The microscopic vesicles can beliposomes. In a preferred embodiment the liposomes are cationicliposomes.

In another aspect, the pharmaceutical composition includes an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof) admixed with a topicalpenetration enhancer. In one embodiment, the topical penetrationenhancer is a fatty acid. The fatty acid can be arachidonic acid, oleicacid, lauric acid, caprylic acid, capric acid, myristic acid, palmiticacid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt.The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid,glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid,sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate,polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable saltthereof

In another embodiment, the penetration enhancer is a chelating agent.The chelating agent can be EDTA, citric acid, a salicyclate, a N-acylderivative of collagen, laureth-9, an N-amino acyl derivative of abeta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g.,an ionic or nonionic surfactant. The surfactant can be sodium laurylsulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether,a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from agroup consisting of unsaturated cyclic ureas, 1-alkyl-alkones,1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents andmixtures thereof. In yet another embodiment the penetration enhancer canbe a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in aform suitable for oral delivery. In one embodiment, oral delivery can beused to deliver an iRNA agent composition to a cell or a region of thegastro-intestinal tract, e.g., small intestine, colon (e.g., to treat acolon cancer), and so forth. The oral delivery form can be tablets,capsules or gel capsules. In one embodiment, the iRNA agent of thepharmaceutical composition modulates expression of a cellular adhesionprotein, modulates a rate of cellular proliferation, or has biologicalactivity against eukaryotic pathogens or retroviruses. In anotherembodiment, the pharmaceutical composition includes an enteric materialthat substantially prevents dissolution of the tablets, capsules or gelcapsules in a mammalian stomach. In a preferred embodiment the entericmaterial is a coating. The coating can be acetate phthalate, propyleneglycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxypropyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a penetration enhancer. The penetration enhancercan be a bile salt or a fatty acid. The bile salt can be ursodeoxycholicacid, chenodeoxycholic acid, and salts thereof. The fatty acid can becapric acid, lauric acid, and salts thereof

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent and a delivery vehicle. In one embodiment, theiRNA agent is (a) is 19-25 nucleotides long, preferably 21-23nucleotides, (b) is complementary to an endogenous target RNA, and,optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) to a cell by a topical route ofadministration. The delivery vehicle can be microscopic vesicles. In oneexample the microscopic vesicles are liposomes. In a preferredembodiment the liposomes are cationic liposomes. In another example themicroscopic vesicles are micelles. In one aspect, the invention featuresa pharmaceutical composition including an iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) in an injectable dosage form. In oneembodiment, the injectable dosage form of the pharmaceutical compositionincludes sterile aqueous solutions or dispersions and sterile powders.In a preferred embodiment the sterile solution can include a diluentsuch as water; saline solution; fixed oils, polyethylene glycols,glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) inoral dosage form. In one embodiment, the oral dosage form is selectedfrom the group consisting of tablets, capsules and gel capsules. Inanother embodiment, the pharmaceutical composition includes an entericmaterial that substantially prevents dissolution of the tablets,capsules or gel capsules in a mammalian stomach. In a preferredembodiment the enteric material is a coating. The coating can be acetatephthalate, propylene glycol, sorbitan monoleate, cellulose acetatetrimellitate, hydroxy propyl methyl cellulose phthalate or celluloseacetate phthalate. In one embodiment, the oral dosage form of thepharmaceutical composition includes a penetration enhancer, e.g., apenetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes an excipient. In one example the excipient ispolyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceuticalcomposition includes a plasticizer. The plasticizer can be diethylphthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethylcitrate.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in arectal dosage form. In one embodiment, the rectal dosage form is anenema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in avaginal dosage form. In one embodiment, the vaginal dosage form is asuppository. In another embodiment, the vaginal dosage form is a foam,cream, or gel.

In one aspect, the invention features a pharmaceutical compositionincluding an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in apulmonary or nasal dosage form. In one embodiment, the iRNA agent isincorporated into a particle, e.g., a macroparticle, e.g., amicrosphere. The particle can be produced by spray drying,lyophilization, evaporation, fluid bed drying, vacuum drying, or acombination thereof. The microsphere can be formulated as a suspension,a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., adouble-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., alarger iRNA agent which can be processed into a sRNA agent, or a DNAwhich encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, or precursor thereof) composition suitable for inhalation by asubject, including: (a) a therapeutically effective amount of a iRNAagent suitable for treating a condition in the subject by inhalation;(b) a pharmaceutically acceptable excipient selected from the groupconsisting of carbohydrates and amino acids; and (c) optionally, adispersibility-enhancing amount of a physiologically-acceptable,water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate canbe selected from the group consisting of monosaccharides, disaccharides,trisaccharides, and polysaccharides. In a preferred embodiment thecarbohydrate is a monosaccharide selected from the group consisting ofdextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. Inanother preferred embodiment the carbohydrate is a disaccharide selectedfrom the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In oneembodiment, the amino acid is a hydrophobic amino acid. In a preferredembodiment the hydrophobic amino acid is selected from the groupconsisting of alanine, isoleucine, leucine, methionine, phenylalanine,proline, tryptophan, and valine. In yet another embodiment the aminoacid is a polar amino acid. In a preferred embodiment the amino acid isselected from the group consisting of arginine, histidine, lysine,cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acidand glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selectedfrom the group consisting of human serum albumin, α-lactalbumin,trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter (MMD) of less than 10 microns.In another embodiment, the spray-dried iRNA agent composition includesparticles having a mass median diameter of less than 5 microns. In yetanother embodiment the spray-dried iRNA agent composition includesparticles having a mass median aerodynamic diameter (MMAD) of less than5 microns.

In certain other aspects, the invention provides kits that include asuitable container containing a pharmaceutical formulation of an iRNAagent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., aprecursor, e.g., a larger iRNA agent which can be processed into a sRNAagent, or a DNA which encodes an iRNA agent, e.g., a double-strandediRNA agent, or sRNA agent, or precursor thereof). In certain embodimentsthe individual components of the pharmaceutical formulation may beprovided in one container. Alternatively, it may be desirable to providethe components of the pharmaceutical formulation separately in two ormore containers, e.g., one container for an iRNA agent preparation, andat least another for a carrier compound. The kit may be packaged in anumber of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

In another aspect, the invention features a device, e.g., an implantabledevice, wherein the device can dispense or administer a composition thatincludes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNAagent, (e.g., a precursor, e.g., a larger iRNA agent which can beprocessed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g.,a double-stranded iRNA agent, or sRNA agent, or precursor thereof),e.g., a iRNA agent that silences an endogenous transcript. In oneembodiment, the device is coated with the composition. In anotherembodiment the iRNA agent is disposed within the device. In anotherembodiment, the device includes a mechanism to dispense a unit dose ofthe composition. In other embodiments the device releases thecomposition continuously, e.g., by diffusion. Exemplary devices includestents, catheters, pumps, artificial organs or organ components (e.g.,artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having thestructure or characteristics of a crystal, i.e., particles ofthree-dimensional structure in which the plane faces intersect atdefinite angles and in which there is a regular internal structure. Thecompositions of the invention may have different crystalline forms.Crystalline forms can be prepared by a variety of methods, including,for example, spray drying.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1: Inhibition of Endogenous ApoM Gene Expression inMice

Apolipoprotein M (ApoM) is a human apolipoprotein predominantly presentin high-density lipoprotein (HDL) in plasma. ApoM is reported to beexpressed exclusively in liver and in kidney (Xu N et al., Biochem JBiol Chem 1999 Oct. 29; 274(44):31286-90). Mouse ApoM is a 21 kDmembrane associated protein, and, in serum, the protein is associatedwith HDL particles. ApoM gene expression is regulated by thetranscription factor hepatocyte nuclear factor 1 alpha (Hnf-1α), asHnf-1α^(−/−) mice are ApoM deficient. In humans, mutations in the HNF-1alpha gene represent a common cause of maturity-onset diabetes of theyoung (MODY).

A variety of test iRNAs were synthesized to target the mouse ApoM gene.This gene was chosen in part because of its high expression levels andexclusive activity in the liver and kidney.

Three different classes of dsRNA agents were synthesized, each classhaving different modifications and features at the 5′ and 3′ ends, seeTable 4.

TABLE 4 Targeted ORF's 5 The23mer: AAGTTTGGGCAGCTCTGCTCT (SEQ ID NO:6708) 19 The23mer: AAGTGGACATACCGATTGACT (SEQ ID NO: 6709) 25 The23mer:AACTCAGAACTGAAGGGCGCC (SEQ ID NO: 6710) 27 The23mer:AAGGGCGCCCAGACATGAAAA (SEQ ID NO: 6711) 3′-UTR (beginning at 645) 42:AAGATAGGAGCCCAGCTTCGA (SEQ ID NO: 6712) Class I 21-nt iRNAs, t,deoxythymidine; p, phosphate pGUUUGGGCAGCUCUGCUCUtt (SEQ ID NO: 6712) #1pAGAGCAGAGCUGCCCAAACtt (SEQ ID NO: 6713) pGUGGACAUACCGAUUGACUtt (SEQ IDNO: 6714) #2 pAGUCAAUCGGUAUGUCCACtt (SEQ ID NO: 6715)pCUCAGAACUGAAGGGCGCCtt (SEQ ID NO: 6716) #3 pGGCGCCCUUCAGUUCUGAGtt (SEQID NO: 6717) pGAUAGGAGCCCAGCUUCGAtt (SEQ ID NO: 6718) #4pUCGAAGCUGGGCUCCUAUCtt (SEQ ID NO: 6719) Class II 21-nt iRNAs, t,deoxythymidine; p, phosphate; ps, thiophosphatepGUUUGGGCAGCUCUGCUCpsUpstpst (SEQ ID NO: 6720) #11pAGAGCAGAGCUGCCCAAApsCpstpst (SEQ ID NO: 6721)pGUGGACAUACCGAUUGACpsUpstpst (SEQ ID NO: 6722) #13pAGUCAAUCGGUAUGUCCApsCpstpst (SEQ ID NO: 6723)pCUCAGAACUGAAGGGCGCpsCpstpst (SEQ ID NO: 6724) #15pGGCGCCCUUCAGUUCUGApsGpstpst (SEQ ID NO: 6725)pGAUAGGAGCCCAGCUUCGpsApstpst (SEQ ID NO: 6726) #17pUCGAAGCUGGGCUCCUAUpsCpstpst (SEQ ID NO: 6727) Class III 23-ntantisense, 21-nt sense, blunt-ended 5′-as GUUUGGGCAGCUCUGCUCUCU (SEQ IDNO: 6728) #19 AGAGAGCAGAGCUGCCCAAACUU (SEQ ID NO: 6729)GUGGACAUACCGAUUGACUGA (SEQ ID NO: 6730) #21 UCAGUCAAUCGGUAUGUCCACUU (SEQID NO: 6731) CUCAGAACUGAAGGGCGCCCA (SEQ ID NO: 6732) #23PUGGGCGCCCUUCAGUUCUGAGUU (SEQ ID NO: 6733) GAUAGGAGCCCAGCUUCGAGU (SEQ IDNO: 6734) #25 ACUCGAAGCUGGGCUCCUAUCUU (SEQ ID NO: 6735)

Class I dsRNAs consisted of 21 nucleotide paired sense and antisensestrands. The sense and antisense strands were each phosphorylated attheir 5′ ends. The double stranded region was 19 nucleotides long andconsisted of ribonucleotides. The 3′ end of each strand created a twonucleotide overhang consisting of two deoxyribonucleotide thymidines.See constructs #1-4 in Table 4.

Class II dsRNAs were also 21 nucleotides long, with a 19 nucleotidedouble strand region. The sense and antisense strands were eachphosphorylated at their 5′ ends. The three 3′ terminal nucleotides ofthe sense and antisense strands were phosphorothioatedeoxyribonucleotides, and the two terminal phosphorothioate thymidineswere unpaired, creating a 3′ overhang region at each end of the iRNAmolecule. See constructs 11, 13, 15, and 17 in Table 4.

Class III dsRNAs included a 23 ribonucleotide antisense strand and a 21ribonucleotide sense strand, to form a construct having a blunt 5′ and a3′ overhang region. See constructs 19, 21, 23, and 25 in Table 4.

Within each of the three classes of iRNAs, the four dsRNA molecules weredesigned to target four different regions of the ApoM transcript. dsRNAs1, 11, and 19 targeted the 5′ end of the open reading frame (ORF).dsRNAs 2, 13, and 21, and 3, 15, and 23, targeted two internal regions(one 5′ proximal and one 3′ proximal) of the ORF, and the 4, 17, and 25iRNA constructs targeted to a region of the 3′ untranslated sequence (3′UTS) of the ApoM mRNA. This is summarized in Table 5.

TABLE 5 iRNA molecules targeted to mouse ApoM iRNA targeted iRNAtargeted iRNA targeted iRNA to 5′ end of to middle ORF to middle ORFtargeted ORF (5′ proximal) (3′ proximal) to 3′UTS Class I 1 2 3 4 ClassII 11 13 15 17 Class III 19 21 23 25

CD1 mice (6-8 weeks old, ˜35 g) were administered one of the test iRNAsin PBS solution. Two hundred micrograms of iRNA in a volume of solutionequal to 10% body weight (˜5.7 mg iRNA/kg mouse) was administered by themethod of high pressure tail vein injection, over a 10-20 sec. timeinterval. After a 24 h recovery period, a second injection was performedusing the same dose and mode of administration as the first injection,and following another 24 h, a third and final injection wasadministered, also using the same dose and mode of administration. Aftera final 24 h recovery, the mouse was sacrificed, serum was collected andthe liver and kidney harvested to assay for an affect on ApoM geneexpression. Expression was monitored by quantitative RT-PCR and Westernblot analyses. This experiment was repeated for each of the iRNAs listedin table 4.

Class I iRNAs did not alter ApoM RNA levels in mice, as indicated byquantitative RT-PCR. This is in contrast to the effect of these iRNAs incultured HepG2 cells. Cells cotransfected with a plasmid expressingexogenous ApoM RNA under a CMV promoter and a class I iRNA demonstrateda 25% or greater reduction in ApoM RNA concentrations as compared tocontrol transfections. The iRNA molecules 1, 2 and 3 each caused a 75%decrease in exogenous ApoM mRNA levels.

Class II iRNAs reduced liver and kidney ApoM mRNA levels by ˜30-85%. TheiRNA molecule “13” elicited the most dramatic reduction in mRNA levels;quantitative RT-PCR indicated a decrease of about 85% in liver tissue.Serum ApoM protein levels were also reduced as was evidenced by Westernblot analysis. The iRNAs 11, 13 and 15, reduced protein levels by about50%, while iRNA 17 had the mildest effect, reducing levels only by˜15-20%.

Class III iRNAs (constructs 19, 21, and 23) reduced serum Apo levels by˜40-50%.

To determine the effect of dosage on iRNA mediated ApoM inhibition, theexperiment described above was repeated with three injections of 50 μgiRNA “11” (˜1.4 mg iRNA/kg mouse). This lower dosage of iRNA resulted ina reduction of serum ApoM levels of about 50%. This is compared with thereduction seen with the 200 μg injections, which reduced serum levels by25-45%. These results indicated the lower dosage amounts of iRNAs wereeffective.

In an effort to increase iRNA uptake by cells, iRNAs were precomplexedwith lipofectamine prior to tail vein injections. ApoM protein levelswere about 50% of wildtype levels in mice injected with iRNA “11” whenthe molecules were preincubated with lipofectamine; ApoM levels werealso about 50% of wildtype when mice were injected with iRNA “11” thatwas not precomplexed with lipofectamine.

These experiments revealed that modified iRNAs can greatly influenceRNAi-mediated gene silencing. As demonstrated herein, modificationsincluding phosphorothioate nucleotides are particularly effective atdecreasing target protein levels.

Example 2: apoB Protein as a Therapeutic Target for Lipid-Based Diseases

Apolipoprotein B (apoB) is a candidate target gene for the developmentof novel therapies for lipid-based diseases.

Methods described herein can be used to evaluate the efficacy of aparticular siRNA as a therapeutic tool for treating lipid metabolismdisorders resulting elevated apoB levels. Use of siRNA duplexes toselectively bind and inactivate the target apoB mRNA is an approach totreat these disorders.

Two approaches:

i) Inhibition of apoB in ex-vivo models by transfecting siRNA duplexeshomologous to human apoB mRNA in a human hepatoma cell line (Hep G2) andmonitor the level of the protein and the RNA using the Western blottingand RT-PCR methods, respectively. siRNA molecules that efficientlyinhibit apoB expression will be tested for similar effects in vivo.

ii) In vivo trials using an apoB transgenic mouse model (apoB100Transgenic Mice, C57BL/6NTac-TgN (APOB100), Order Model #'s: 1004-T(hemizygotes), B6 (control)). siRNA duplexes are designed to targetapoB-100 or CETP/apoB double transgenic mice which express bothcholesteryl ester transfer protein (CETP) and apoB. The effect of thesiRNA on gene expression in vivo can be measured by monitoring theHDL/LDL cholesterol level in serum. The results of these experimentswould indicate the therapeutic potential of siRNAs to treat lipid-baseddiseases, including hypercholesterolemia, HDL/LDL cholesterol imbalance,familial combined hyperlipidemia, and acquired hyperlipidemia.

Background

Fats, in the form of triglycerides, are ideal for energy storage becausethey are highly reduced and anhydrous. An adipocyte (or fat cell)consists of a nucleus, a cell membrane, and triglycerides, and itsfunction is to store triglycerides.

The lipid portion of the human diet consists largely of triglyceridesand cholesterol (and its esters). These must be emulsified and digestedto be absorbed. Specifically, fats (triacylglycerols) are ingested. Bile(bile acids, salts, and cholesterol), which is made in the liver, issecreted by the gall bladder. Pancreatic lipase digests thetriglycerides to fatty acids, and also digests di-, andmono-acylglycerols, which are absorbed by intestinal epithelial cellsand then are resynthesized into triacylglycerols once inside the cells.These triglycerides and some cholesterols are combined withapolipoproteins to produce chylomicrons. Chylomicrons consist ofapproximately 95% triglycerides. The chylomicrons transport fatty acidsto peripheral tissues. Any excess fat is stored in adipose tissue.

Lipid transport and clearance from the blood into cells, and from thecells into the blood and the liver, is mediated by the lipoproteintransport proteins. This class of approximately 17 proteins can bedivided into three groups: Apolipoproteins, lipoprotein processingproteins, and lipoprotein receptors.

Apolipoproteins coat lipoprotein particles, and include the A-I, A-II,A-IV, B, CII, CIII, D, E, Apo(a) proteins. Lipoprotein processingproteins include lipoprotein lipase, hepatic lipase, lecithincholesterol acyltransferase and cholesterol ester transfer protein.Lipoprotein receptors include the low density lipoprotein (LDL)receptor, chylomicron-remnant receptor (the LDL receptor like protein orLDL receptor related protein—LRP) and the scavenger receptor.

Lipoprotein Metabolism Since the triglycerides, cholesterol esters, andcholesterol absorbed into the small intestine are not soluble in aqueousmedium, they must be combined with suitable proteins (apolipoproteins)in order to prevent them from forming large oil droplets. The resultinglipoproteins undergo a type of metabolism as they pass through thebloodstream and certain organs (notably the liver).

Also synthesized in the liver is high density lipoprotein (HDL), whichcontains the apoproteins A-1, A-2, C-1, and D; HDL collects cholesterolfrom peripheral tissues and blood vessels and returns it to the liver.LDL is taken up by specific cell surface receptors into an endosome,which fuses with a lysosome where cholesterol ester is converted to freecholesterol. The apoproteins (including apo B-100) are digested to aminoacids. The receptor protein is recycled to the cell membrane.

The free cholesterol formed by this process has two fates. First, it canmove to the endoplasmic reticulum (ER), where it can inhibit HMG-CoAreductase, the synthesis of HMG-CoA reductase, and the synthesis of cellsurface receptors for LDL. Also in the ER, cholesterol can speed up thedegradation of HMG-CoA reductase. The free cholesterol can also beconverted by acyl-CoA and acyl transferase (ACAT) to cholesterol esters,which form oil droplets.

ApoB is the major apolipoprotein of chylomicrons of very low densitylipoproteins (VLDL, which carry most of the plasma triglyceride) and lowdensity lipoprotein (LDL, which carry most of the plasma cholesterol).ApoB exists in human plasma in two isoforms, apoB-48 and apoB-100.

ApoB-100 is the major physiological ligand for the LDL receptor. TheApoB precursor has 4563 amino acids, and the mature apoB-100 has 4536amino acid residues. The LDL-binding domain of ApoB-100 is proposed tobe located between residues 3129 and 3532. ApoB-100 is synthesized inthe liver and is required for the assembly of very low densitylipoproteins VLDL and for the preparation of apoB-100 to transporttriglycerides (TG) and cholesterol from the liver to other tissues.ApoB-100 does not interchange between lipoprotein particles, as do theother lipoproteins, and it is found in IDL and LDL particles. After theremoval of apolipoproteins A, E and C, apoB is incorporation into VLDLby hepatocytes. ApoB-48 is present in chylomicrons and plays anessential role in the intestinal absorption of dietary fats. ApoB-48 issynthesized in the small intestine. It comprises the N-terminal 48% ofapoB-100 and is produced by a posttranscriptional apoB-100 mRNA editingevent at codon 2153 (C to U). This editing event is a product of theapoBEC-lb enzyme, which is expressed in the intestine. This editingevent creates a stop codon instead of a glutamine codon, and thereforeapoB-48, instead of apoB-100 is expressed in the intestine (apoB-100 isexpressed in the liver).

There is also strong evidence that plasma apoB levels may be a betterindex of the risk of coronary artery disease (CAD) than total or LDLcholesterol levels. Clinical studies have demonstrated the value ofmeasuring apoB in hypertriglyceridemic, hypercholesterolemic andnormalipidemic subjects.

TABLE 6 Reference Range Lipid level in the Blood Lipid Range (mmols/L)Plasma Cholesterol 3.5-6.5 Low density lipoprotein 1.55-4.4  Very lowdensity lipoprotein 0.128-0.645 High density lipoprotein/triglycerides0.5-2.1 Total lipid 4.0-10 g/L

Molecular Genetics of Lipid Metabolism in Both Humans and Induced MutantMouse Models

Elevated plasma levels of LDL and apoB are associated with a higher riskfor atherosclerosis and coronary heart disease, a leading cause ofmortality. ApoB is the mandatory constituent of LDL particles. Inaddition to its role in lipoprotein metabolism, apoB has also beenimplicated as a factor in male infertility and fetal development.Furthermore, two quantitative trait loci regulating plasma apoB levelshave been discovered, through the use of transgenic mouse models. Futureexperiments will facilitate the identification of human orthologousgenes encoding regulators of plasma apoB levels. These loci arecandidate therapeutic targets for human disorders characterized byaltered plasma apoB levels. Such disorders include non-apoB linkedhypobetalipoproteinemia and familial combined hyperlipidemia. Theidentification of these genetic loci would also reveal possible newpathways involved in the regulation of apoB secretion, potentiallyproviding novel sites for pharmacological therapy.

Diseases and Clinical Pharmacology

Familial combined hyperlipemia (FCHL) affects an estimated one in 10Americans. FCHL can cause premature heart disease.

Familial Hypercholesterolemia (High Level of apo B)

A common genetic disorder of lipid metabolism. Familialhypercholesterolemia is characterized by elevated serum TC inassociation with xanthelasma, tendon and tuberous xanthomas, acceleratedatherosclerosis, and early death from myocardial infarction (MI). It iscaused by absent or defective LDL cell receptors, resulting in delayedLDL clearance, an increase in plasma LDL levels, and an accumulation ofLDL cholesterol in macrophages over joints and pressure points, and inblood vessels.

Atherosclerosis (High Level of apo B)

Atherosclerosis develops as a deposition of cholesterol and fat in thearterial wall due to disturbances in lipid transport and clearance fromthe blood into cells and from the cells to blood and the liver.

Clinical studies have demonstrated that elevation of total cholesterol(TC), low-density lipoprotein cholesterol (LDL-C) and apoB-100 promotehuman atherosclerosis. Similarly, decreased levels of high-densitylipoprotein cholesterol (HDL-C) are associated with the development ofatherosclerosis.

ApoB may be factor in the genetic cause of high cholesterol.

The Risk of Coronary Artery Disease (CAD) (High Level of apo B)

Cardiovascular disease, including coronary heart disease and stroke, isa leading cause of death and disability. The major risk factors includeage, gender, elevated low-density lipoprotein cholesterol blood levels,decreased high-density lipoprotein cholesterol levels, cigarettesmoking, hypertension, and diabetes. Emerging risk factors includeelevated lipoprotein (a), remnant lipoproteins, and C reactive protein.Dietary intake, physical activity and genetics also impactcardiovascular risk. Hypertension and age are the major risk factors forstroke.

Abetalipoproteinemia, an inherited human disease characterized by anear-complete absence of apoB-containing lipoproteins in the plasma, iscaused by mutations in the gene for microsomal triglyceride transferprotein (MTP).

Model for Human Atherosclerosis (Lipoprotein a Transgenic Mouse)

Numerous studies have demonstrated that an elevated plasma level oflipoprotein(a) (Lp(a)) is a major independent risk factor for coronaryheart disease (CHD). Current therapies, however, have little or noeffect on apo(a) levels and the homology between apo(a) and plasminogenpresents barriers to drug development. Lp(a) particles consist of apo(a)and apoB-100 proteins, and they are found only in primates and thehedgehog. The development of LPA transgenic mouse requires the creationof animals that express both human apoB and apo(a) transgenes to achieveassembly of LP(a). An atherosclerosis mouse model would facilitate thestudy of the disease process and factors influencing it, and furtherwould facilitate the development of therapeutic or preventive agents.There are several strategies for gene-oriented therapy. For example, themissing or non-functional gene can be replaced, or unwanted geneactivity can be inhibited.

Model for Lipid Metabolism and Atherosclerosis

DNX Transgenic Sciences has demonstrated that both CETP/ApoB and ApoBtransgenic mice develop atherosclerotic plaques.

Model for apoB-100 Overexpression

The apoB-100 transgenic mice express high levels of human apoB-100. Theyconsequently demonstrate elevated serum levels of LDL cholesterol. After6 months on a high-fat diet, the mice develop significant foam cellaccumulation under the endothelium and within the media, as well ascholesterol crystals and fibrotic lesions.

Model for Cholesteryl Ester Transfer Protein Over Expression

The apoB-100 transgenic mice express the human enzyme, CETP, andconsequently demonstrate a dramatically reduced level of serum HDLcholesterol.

Model for apoB-100 and CETP Overexpression

The apoB-100 transgenic mice express both CETP and apoB-100, resultingin mice with a human like serum HDL/LDL distribution. Following 6 monthson a high-fat diet these mice develop significant foam cell accumulationunderlying the endothelium and within the media, as well as cholesterolcrystals and fibrotic lesions.

ApoB100 Transgenic Mice (Order Model #'s: 1004-T (Hemizygotes), B6(Control))

These mice express high levels of human apoB-100, resulting in mice withelevated serum levels of LDL cholesterol. These mice are useful inidentifying and evaluating compounds to reduce elevated levels of LDLcholesterol and the risk of atherosclerosis. When fed a high fatcholesterol diet, these mice develop significant foam cell accumulationunderly the endothelium and within the media, and have significantlymore complex atherosclerotic lesions than control animals.

Double Transgenic Mice, CETP/ApoB100 (Order Model #: 1007-TT)

These mice express both CETP and apoB-100, resulting in a human-likeserum HDL/LDL distribution. These mice are useful for evaluatingcompounds to treat hypercholesterolemia or HDL/LDL cholesterol imbalanceto reduce the risk of developing atherosclerosis. When fed a high fathigh cholesterol diet, these mice develop significant foam cellaccumulation underlying the endothelium and within the media, and havesignificantly more complex atherosclerotic lesions than control animals.

ApoE Gene Knockout Mouse

Homozygous apoE knockout mice exhibit strong hypercholesterolemia,primarily due to elevated levels of VLDL and IDL caused by a defect inlipoprotein clearance from plasma. These mice develop atheroscleroticlesions which progress with age and resemble human lesions (Zhang etal., Science 258:46-71, 1992; Plump et al., Cell 71:343-353, 1992;Nakashima et al., Arterioscler Thromp. 14:133-140, 1994; Reddick et al.,Arterioscler Tromb. 14:141-147, 1994). These mice are a promising modelfor studying the effect of diet and drugs on atherosclerosis.

Low density lipoprotein receptor (LDLR) mediates lipoprotein clearancefrom plasma through the recognition of apoB and apoE on the surface oflipoprotein particles. Humans, who lack or have a decreased number ofthe LDL receptors, have familial hypercholesterolemia and develop CHD atan early age.

ApoE Knockout Mice (Order Model #: APOE-M)

The apoE knockout mouse was created by gene targeting in embryonic stemcells to disrupt the apoE gene. ApoE, a glycoprotein, is a structuralcomponent of very low density lipoprotein (VLDL) synthesized by theliver and intestinally synthesized chylomicrons. It is also aconstituent of a subclass of high density lipoproteins (HDLs) involvedin cholesterol transport activity among cells. One of the most importantroles of apoE is to mediate high affinity binding of chylomicrons andVLDL particles that contain apoE to the low density lipoprotein (LDL)receptor. This allows for the specific uptake of these particles by theliver which is necessary for transport preventing the accumulation inplasma of cholesterol-rich remnants. The homozygous inactivation of theapoE gene results in animals that are devoid of apoE in their sera. Themice appear to develop normally, but they exhibit five times the normalserum plasma cholesterol and spontaneous atherosclerotic lesions. Thisis similar to a disease in people who have a variant form of the apoEgene that is defective in binding to the LDL receptor and are at riskfor early development of atherosclerosis and increased plasmatriglyceride and cholesterol levels. There are indications that apoE isalso involved in immune system regulation, nerve regeneration and muscledifferentiation. The apoE knockout mice can be used to study the role ofapoE in lipid metabolism, atherogenesis, and nerve injury, and toinvestigate intervention therapies that modify the atherogenic process.

Apoe4 Targeted Replacement Mouse (Order Model #: 001549-M)

ApoE is a plasma protein involved in cholesterol transport, and thethree human isoforms (E2, E3, and E4) have been associated withatherosclerosis and Alzheimer's disease. Gene targeting of 129 ES cellswas used to replace the coding sequence of mouse apoE with human APOE4without disturbing the murine regulatory sequences. The E4 isoformoccurs in approximately 14% of the human population and is associatedwith increased plasma cholesterol and a greater risk of coronary arterydisease. The Taconic apoE4 Targeted Replacement model has normal plasmacholesterol and triglyceride levels, but altered quantities of differentplasma lipoprotein particles. This model also has delayed plasmaclearance of cholesterol-rich lipoprotein particles (VLDL), with onlyhalf the clearance rate seen in the apoE3 Targeted Replacement model.Like the apoE3 model, the apoE4 mice develop altered plasma lipoproteinvalues and atherosclerotic plaques on an atherogenic diet. However, theatherosclerosis is more severe in the apoE4 model, with larger plaquesand cholesterol apoE and apoB-48 levels twice that seen in the apoE3model. The Taconic apoE4 Targeted Replacement model, along with theapoE2 and apoE3 Targeted Replacement Mice, provide an excellent tool forin vivo study of the human apoE isoforms.

CETP Transgenic Mice (Order Model #: 1003-T)

These animals express the human plasma enzyme, CETP, resulting in micewith a dramatic reduction in serum HDL cholesterol. The mice can beuseful in identifying and evaluating compounds that increase the levelsof HDL cholesterol for reducing the risk of developing atherosclerosis

Transgene/Promoter: Human Apolipoprotein A-I

These mice produce mouse HDL cholesterol particles that contain humanapolipoprotein A-I. Transgenic expression is life-long in both sexes(Biochemical Genetics and Metabolism Laboratory, Rockefeller University,NY City).

A Mouse Model for Abetalipoproteinemia

Abetalipoproteinemia, an inherited human disease characterized by anear-complete absence of apoB-containing lipoproteins in the plasma, iscaused by mutations in the gene for microsomal triglyceride transferprotein (MTP). Gene targeting was used to knock out the mouse MTP gene(Mttp). In heterozygous knockout mice (Mttp^(+/−)), the MTP mRNA,protein, and activity levels were reduced by 50% in both liver andintestine. Recent studies with heterozygous MTP knockout mice havesuggested that half-normal levels of MTP in the liver reduce apoBsecretion. They hypothesized that reduced apoB secretion in the settingof half-normal MTP levels might be caused by a reduced MTP:apoB ratio inthe endoplasmic reticulum, which would reduce the number of apoB-MTPinteractions. If this hypothesis were true, half-normal levels of MTPmight have little impact on lipoprotein secretion in the setting ofhalf-normal levels of apoB synthesis (since the ratio of MTP to apoBwould not be abnormally low) and might cause an exaggerated reduction inlipoprotein secretion in the setting of apoB overexpression (since theratio of MTP to apoB would be even lower). To test this hypothesis, theyexamined the effects of heterozygous MTP deficiency on apoB metabolismin the setting of normal levels of apoB synthesis, half-normal levels ofapoB synthesis (heterozygous Apob deficiency), and increased levels ofapoB synthesis (transgenic overexpression of human apoB). Contrary totheir expectations, half-normal levels of MTP reduced plasma apoB-100levels to the same extent (˜25-35%) at each level of apoB synthesis. Inaddition, apoB secretion from primary hepatocytes was reduced to acomparable extent at each level of apoB synthesis. Thus, these resultsindicate that the concentration of MTP within the endoplasmic reticulum,rather than the MTP:apoB ratio, is the critical determinant oflipoprotein secretion. Finally, heterozygosity for an apoB knockoutmutation was found to lower plasma apoB-100 levels more thanheterozygosity for an MTP knockout allele. Consistent with that result,hepatic triglyceride accumulation was greater in heterozygous apoBknockout mice than in heterozygous MTP knockout mice. Cre/loxPtissue-specific recombination techniques were also used to generateliver-specific Mttp knockout mice. Inactivation of the Mttp gene in theliver caused a striking reduction in very low density lipoprotein (VLDL)triglycerides and large reductions in both VLDL/low density lipoproteins(LDL) and high density lipoprotein cholesterol levels. Histologicstudies in liver-specific knockout mice revealed moderate hepaticsteatosis. Currently being tested is the hypothesis that accumulation oftriglycerides in the liver renders the liver more susceptible to injuryby a second insult (e.g., lipopolysaccharide).

Human apo B (Apolipoprotein B) Transgene Mice Show apo B Locus May havea Causative Role Male Infertility

The fertility of apoB (apolipoprotein B) (+/−) mice was recorded duringthe course of backcrossing (to C57BL/6J mice) and test mating. Noapparent fertility problem was observed in female apoB (+/−) andwild-type female mice, as was documented by the presence of vaginalplugs in female mice. Although apoB (+/−) mice mated normally, only 40%of the animals from the second backcross generation produced anyoffspring within the 4-month test period. Of the animals that producedprogeny, litters resulted from <50% of documented matings. In contrast,all wild-type mice (6/6—i.e., 100%) tested were fertile. These datasuggest genetic influence on the infertility phenotype, as a smallnumber of male heterozygotes were not sterile. Fertilization in vivo wasdramatically impaired in male apoB (+/−) mice. 74% of eggs examined werefertilized by the sperm from wild-type mice, whereas only 3% of eggsexamined were fertilized by the sperm from apoB (+/−) mice. The spermcounts of apoB (+/−) mice were mildly but significantly reduced comparedwith controls. However, the percentage of motile sperm was markedlyreduced in the apoB (+/−) animals compared with that of the wild-typecontrols. Of the sperm from apoB (+/−) mice, 20% (i.e., 4.9% of theinitial 20% motile sperm) remained motile after 6 hr of incubation,whereas 45% (i.e., 33.6% of the initial 69.5%) of the motile spermretained motility in controls after this time. In vitro fertilizationyielded no fertilized eggs in three attempts with apo B (+/−) mice,while wild-type controls showed a fertilization rate of 53%. However,sperm from apoB (+/−) mice fertilized 84% of eggs once the zonapellucida had been removed. Numerous sperm from apoB (+/−) mice wereseen binding to zona-intact eggs. However, these sperm lost theirmotility when observed 4-6 hours after binding, showing that sperm fromapoB (+/−) mice were unable to penetrate the zona pellucida but that theinteraction between sperm and egg was probably not direct. Sperm bindingto zona-free oocytes was abnormal. In the apoB (+/−) mice, sperm bindingdid not attenuate, even after pronuclei had clearly formed, suggestingthat apoB deficiency results in abnormal surface interaction between thesperm and egg.

Knockout of the mouse apoB gene resulted in embryonic lethality inhomozygotes, protection against diet-induced hypercholesterolemia inheterozygotes, and developmental abnormalities in mice.

Model of Insulin Resistance, Dyslipidemia & Overexpression of Human apoB

It was shown that the livers of apoB mice assemble and secrete increasednumbers of VLDL particles.

Example 3. Treatment of Diabetes Type-2 with iRNA Introduction

The regulation of hepatic gluconeogenesis is an important process in theadjustment of the blood glucose level. Pathological changes in theglucose production of the liver are a central characteristic intype-2-diabetes. For example, the fasting hyperglycemia observed inpatients with type-2-diabetes reflects the lack of inhibition of hepaticgluconeogenesis and glycogenolysis due to the underlying insulinresistance in this disease. Extreme conditions of insulin resistance canbe observed for example in mice with a liver-specific insulin receptorknockout (‘LIRKO’). These mice have an increased expression of the tworate-limiting gluconeogenic enzymes, phosphoenolpyruvate carboxykinase(PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase).Insulin is known to repress both PEPCK and G6Pase gene expression at thetranscriptional level and the signal transduction involved in theregulation of G6Pase and PEPCK gene expression by insulin is only partlyunderstood. While PEPCK is involved in a very early step of hepaticgluconeogenesis (synthesis of phosphoenolpyruvate from oxaloacetate),G6Pase catalyzes the terminal step of both, gluconeogenesis andglycogenolysis, the cleavage of glucose-6-phosphate into phosphate andfree glucose, which is then delivered into the blood stream.

The pharmacological intervention in the regulation of expression ofPEPCK and G6Pase can be used for the treatment of the metabolicaberrations associated with diabetes. Hepatic glucose production can bereduced by an iRNA-based reduction of PEPCK and G6Pase enzymaticactivity in subjects with type-2-diabetes.

Targets for iRNA

Glucose-6-Phosphatase (G6Pase)

G6Pase mRNA is expressed principally in liver and kidney, and in loweramounts in the small intestine. Membrane-bound G6Pase is associated withthe endoplasmic reticulum. Low activities have been detected in skeletalmuscle and in astrocytes as well.

G6Pase catalyzes the terminal step in gluconeogenesis andglycogenolysis. The activity of the enzyme is several fold higher indiabetic animals and probably in diabetic humans. Starvation anddiabetes cause a 2-3-fold increase in G6Pase activity in the liver and a2-4-fold increase in G6Pase mRNA.

Phosphoenolpyruvate Carboxykinase (PEPCK)

Overexpression of PEPCK in mice results in symptoms of type-2-diabetesmellitus. PEPCK overexpression results in a metabolic pattern thatincreases G6Pase mRNA and results in a selective decrease in insulinreceptor substrate (IRS)-2 protein, decreased phosphatidylinositol3-kinase activity, and reduced ability of insulin to suppressgluconeogenic gene expression.

TABLE 7 Other targets to inhibit hepatic glucose production TargetComment FKHR good evidence for antidiabetic phenotype (Nakae et al., NatGenetics 32: 245(2002) Glucagon Glucagon receptor Glycogen phosphorylasePGC-1 (PPAR-Gamma regulates the cAMP response (and Coactivator) probablythe PKB/FKHR-regulation) on PEPCK/G6Pase Fructose-1,6-bisphosphataseGlucose-6-phospate translocator Glucokinase inhibitory regulatoryprotein

Materials and Methods

Animals: BKS.Cg-m+/+ Lepr db mice, which contain a point mutation in theleptin receptor gene are used to examine the efficacy of iRNA for thetargets listed above.

BKS.Cg-m+/+ Lepr db are available from the Jackson Laboratory (StockNumber 000642). These animals are obese at 3-4 weeks after birth, showelevation of plasma insulin at 10 to 14 days, elevation of blood sugarat 4 to 8 weeks, and uncontrolled rise in blood sugar. Exogenous insulinfails to control blood glucose levels and gluconeogenic activityincreases.

The following numbers of male animals (age>12 weeks) would ideally betested with the following iRNAs:

-   -   PEPCK, 2 sequences, 5 animals per sequence    -   G6Pase, 2 sequences, 5 animals per sequence    -   1 nonspecific sequence, 5 animals    -   1 control group (only injected, no siRNA), 5 animals    -   1 control group (not injected, no siRNA), 5 animals        Reagents: Necessary reagents would ideally include a Glucometer        Elite XL (Bayer, Pittsburgh, Pa.) for glucose quantification,        and an Insulin Radioimmunoassay (RIA) kit (Amersham, Piscataway,        N.J.) for insulin quanitation

Assays:

G6P enzyme assays and PEPCK enzyme assays are used to measure theactivity of the enzymes. Northern blotting is used to detect levels ofG6Pase and PEPCK mRNA. Antibody-based techniques (e.g., immunoblotting,immunofluorescence) are used to detect levels of G6Pase and PEPCKprotein. Glycogen staining is used to detect levels of glycogen in theliver. Histological analysis is performed to analyze tissues.

Gene Information:

G6Pase GenBank® No.: NM_008061, Mus musculus glucose-6-phosphatase,catalytic (G6pc), mRNA 1 . . . 2259, ORF 83 . . . 1156;GenBank® No: U00445, Mus musculus glucose-6-phosphatase mRNA, completecds 1 . . . 2259, ORF 83 . . . 1156

GenBank® No: BC013448 PEPCK

GenBank® No: NM_011044, Mus musculus phosphoenolpyruvate carboxykinase1, cytosolic (Pck1), mRNA. 1 . . . 2618, ORF 141 . . . 2009

GenBank® No: AF009605.1

Administration of iRNA:

iRNA corresponding to the genes described above would be administered tomice with hydrodynamic injection. One control group of animals would betreated with Metformin as a positive control for reduction in hepaticglucose levels.

Experimental Protocol

Mice would be housed in a facility in which there is light from 7:00 AMto 7:00 PM. Mice would be fed ad libidum from 7:00 PM to 7:00 AM andfast from 7:00 AM to 7:00 PM.

Day 0: 7:00 PM: Approximately 100 μl blood would be drawn from the tail.Serum would be isolated to measure glucose, insulin, HbA1c (EDTA-blood),glucagon, FFAs, lactate, corticosterone, serum triglycerides.Day 1-7: Blood glucose would be measured daily at 8:00 AM and 6:00 PM(approx. 3-5 μl; measured with a Haemoglucometer)Day 8: Blood glucose would be measured daily at 8:00 AM and 6:00 PM.iRNA would be injected between 10:00 AM and 2:00 PMDay 9-20: Blood glucose would be measured daily at 8:00 AM and 6:00 PM.Day 21: Mice would be sacrificed after 10 hours of fasting.Blood would be isolated. Glucose, insulin, HbA1c (EDTA-blood), glucagon,FFAs, lactate, corticosterone, serum triglycerides would be measured.Liver tissue would be isolated for histology, protein assays, RNAassays, glycogen quantitation, and enzyme assays.

Example 4: Inhibition of Glucose-6-Phosphatase iRNA In Vivo

iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used toexamine the effects of inhibition of G6P expression on glucosemetabolism in vivo.

Female mice, 10 weeks of age, strain BKS.Cg-m +/+ Lepr db (The JacksonLaboratory) were used for in vivo analysis of enzymes of the hepaticglucose production. Mice were housed under conditions where it was lightfrom 6:30 am to 6:30 pm. Mice were fed (ad libidum) during the nightperiod and fasted during the day period.

On day 1, approximately 100 μl of blood was collected from test animalsby puncturing the retroorbital plexus. On days 1-7, blood glucose wasmeasured in blood obtained from tail veins (approximately 3-5 μl) usinga Glucometer (Elite XL, Bayer). Blood glucose was sampled daily at 8 amand 6 pm.

On day 7 at approximately 2 pm, GL3 plasmid (10 μg) and siRNAs (100 μgG6Pase specific, Renilla nonspecific or no siRNA control) were deliveredto animals using hydrodynamic coinjection.

On day 8, GL3 expression was analyzed by injection of luceferin (3 mg)after anaesthesia with avertin and imaging. This was done to control forsuccessful hydrodynamic delivery.

On days 8-10, blood glucose was measured in blood obtained from tailveins (approximately 3-5 ml) using a Glucometer (Elite XL, Bayer).

On day 10, mice were sacrificed after 10 hours of fasting. Blood andliver were isolated from sacrificed animals.

Results: Coinjection of GL3 plasmid and G6Pase iRNA (G6P4) reduced bloodglucose levels for the short term. Coinjection of GL3 plasmid andRenilla nonspecific iRNA had no effect on blood glucose levels.

Example 5: Selected Palindromic Sequences

Tables 8-13 below provide selected palindromic sequences from thefollowing genes: human ApoB, human glucose-6-phosphatase, ratglucose-6-phosphatase, β-catenin, and hepatitis C virus (HCV).

TABLE 8 Selected palindromic sequences from human ApoB Start End StartEnd Source Index Index Match Index Index # B SEQ ID NO: 1ggccattccagaagggaag 509 528 SEQ ID NO: 1004 cttccgttctgtaatggcc 57955814 1 9 SEQ ID NO: 2 tgccatctcgagagttcca 4099 4118 SEQ ID NO: 1005tggaactctctccatggca 10876 10895 1 8 SEQ ID NO: 3 catgtcaaacactttgtta7056 7075 SEQ ID NO: 1006 taacaaattccttgacatg 7358 7377 1 8 SEQ ID NO: 4tttgttataaatcttattg 7068 7087 SEQ ID NO: 1007 caataagatcaatagcaaa 89909009 1 8 SEQ ID NO: 5 tctggaaaagggtcatgga 8880 8899 SEQ ID NO: 1008tccatgtcccatttacaga 11356 11375 1 8 SEQ ID NO: 6 cagctcttgttcaggtcca10900 10919 SEQ ID NO: 1009 tggacctgcaccaaagctg 13952 13971 1 8 SEQ IDNO: 7 ggaggttccccagctctgc 356 375 SEQ ID NO: 1010 gcagccctgggaaaactcc6447 6466 1 7 SEQ ID NO: 8 ctgttttgaagactctcca 1081 1100 SEQ ID NO: 1011tggagggtagtcataacag 10327 10346 1 7 SEQ ID NO: 9 agtggctgaaacgtgtgca1297 1316 SEQ ID NO: 1012 tgcagagctttctgccact 13508 13527 1 7 SEQ ID NO:10 ccaaaatagaagggaatct 2068 2087 SEQ ID NO: 1013 agattcctttgccttttgg4000 4019 1 7 SEQ ID NO: 11 tgaagagaagattgaattt 3620 3639 SEQ ID NO:1014 aaattctcttttcttttca 9212 9231 1 7 SEQ ID NO: 12 agtggtggcaacaccagca4230 4249 SEQ ID NO: 1015 tgctagtgaggccaacact 10649 10668 1 7 SEQ ID NO:13 aaggctccacaagtcatca 5950 5969 SEQ ID NO: 1016 tgatgatatctggaacctt10724 10743 1 7 SEQ ID NO: 14 gtcagccaggtttatagca 7725 7744 SEQ ID NO:1017 tgctaagaaccttactgac 7781 7800 1 7 SEQ ID NO: 15 tgatatctggaaccttgaa10727 10746 SEQ ID NO: 1018 ttcactgttcctgaaatca 7863 7882 1 7 SEQ ID NO:16 gtcaagttgagcaatttct 13423 13442 SEQ ID NO: 1019 agaaaaggcacaccttgac11072 11091 1 7 SEQ ID NO: 17 atccagatggaaaagggaa 13480 13499 SEQ ID NO:1020 ttccaatttccctgtggat 3680 3699 1 7 SEQ ID NO: 18 atttgtttgtcaaagaagt4543 4562 SEQ ID NO: 1021 acttcagagaaatacaaat 11401 11420 4 6 SEQ ID NO:19 ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1022 ccagacttccgtttaccag 82358254 2 6 SEQ ID NO: 20 accaggaggttcttcttca 1729 1748 SEQ ID NO: 1023tgaagtgtagtctcctggt 5089 5108 2 6 SEQ ID NO: 21 aaagaagttctgaaagaat 19561975 SEQ ID NO: 1024 attccatcacaaatccttt 9661 9680 2 6 SEQ ID NO: 22gctacagcttatggctcca 3570 3589 SEQ ID NO: 1025 tggatctaaatgcagtagc 1162311642 2 6 SEQ ID NO: 23 atcaatattgatcaatttg 6414 6433 SEQ ID NO: 1026caaagaagtcaagattgat 4553 4572 2 6 SEQ ID NO: 24 gaattatcttttaaaacat 73267345 SEQ ID NO: 1027 atgtgttaacaaaatattc 11494 11513 2 6 SEQ ID NO: 25cgaggcccgcgctgctggc 130 149 SEQ ID NO: 1028 gccagaagtgagatcctcg 35073526 1 6 SEQ ID NO: 26 acaactatgaggctgagag 271 290 SEQ ID NO: 1029ctctgagcaacaaatttgt 10309 10328 1 6 SEQ ID NO: 27 gctgagagttccagtggag282 301 SEQ ID NO: 1030 ctccatggcaaatgtcagc 10885 10904 1 6 SEQ ID NO:28 tgaagaaaaccaagaactc 448 467 SEQ ID NO: 1031 gagtcattgaggttcttca 49294948 1 6 SEQ ID NO: 29 cctacttacatcctgaaca 558 577 SEQ ID NO: 1032tgttcataagggaggtagg 12766 12785 1 6 SEQ ID NO: 30 ctacttacatcctgaacat559 578 SEQ ID NO: 1033 atgttcataagggaggtag 12765 12784 1 6 SEQ ID NO:31 gagacagaagaagccaagc 615 634 SEQ ID NO: 1034 gcttggttttgccagtctc 24592478 1 6 SEQ ID NO: 32 cactcactttaccgtcaag 671 690 SEQ ID NO: 1035cttgaacacaaagtcagtg 6000 6019 1 6 SEQ ID NO: 33 ctgatcagcagcagccagt 822841 SEQ ID NO: 1036 actgggaagtgcttatcag 5237 5256 1 6 SEQ ID NO: 34actggacgctaagaggaag 854 873 SEQ ID NO: 1037 cttccccaaagagaccagt 28902909 1 6 SEQ ID NO: 35 agaggaagcatgtggcaga 865 884 SEQ ID NO: 1038tctggcatttactttctct 5921 5940 1 6 SEQ ID NO: 36 tgaagactctccaggaact 10871106 SEQ ID NO: 1039 agttgaaggagactattca 7216 7235 1 6 SEQ ID NO: 37ctctgagcaaaatatccag 1121 1140 SEQ ID NO: 1040 ctggttactgagctgagag 11611180 1 6 SEQ ID NO: 38 atgaagcagtcacatctct 1189 1208 SEQ ID NO: 1041agagctgccagtccttcat 10016 10035 1 6 SEQ ID NO: 39 ttgccacagctgattgagg1209 1228 SEQ ID NO: 1042 cctcctacagtggtggcaa 4222 4241 1 6 SEQ ID NO:40 agctgattgaggtgtccag 1216 1235 SEQ ID NO: 1043 ctggattccacatgcagct11847 11866 1 6 SEQ ID NO: 41 tgctccactcacatcctcc 1278 1297 SEQ ID NO:1044 ggaggctttaagttcagca 7601 7620 1 6 SEQ ID NO: 42 tgaaacgtgtgcatgccaa1303 1322 SEQ ID NO: 1045 ttgggagagacaagtttca 6500 6519 1 6 SEQ ID NO:43 gacattgctaattacctga 1503 1522 SEQ ID NO: 1046 tcagaagctaagcaatgtc7232 7251 1 6 SEQ ID NO: 44 ttcttcttcagactttcct 1738 1757 SEQ ID NO:1047 aggagagtccaaattagaa 8498 8517 1 6 SEQ ID NO: 45 ccaatatcttgaactcaga1903 1922 SEQ ID NO: 1048 tctgaattcattcaattgg 6485 6504 1 6 SEQ ID NO:46 aaagttagtgaaagaagtt 1946 1965 SEQ ID NO: 1049 aactaccctcactgccttt2132 2151 1 6 SEQ ID NO: 47 aagttagtgaaagaagttc 1947 1966 SEQ ID NO:1050 gaacctctggcatttactt 5916 5935 1 6 SEQ ID NO: 48 aaagaagttctgaaagaat1956 1975 SEQ ID NO: 1051 attctctggtaactacttt 5482 5501 1 6 SEQ ID NO:49 tttggctataccaaagatg 2322 2341 SEQ ID NO: 1052 catcttaggcactgacaaa4997 5016 1 6 SEQ ID NO: 50 tgttgagaagctgattaaa 2381 2400 SEQ ID NO:1053 tttagccatcggctcacca 5700 5719 1 6 SEQ ID NO: 51 caggaagggctcaaagaat2561 2580 SEQ ID NO: 1054 attcctttaacaattcctg 9492 9511 1 6 SEQ ID NO:52 aggaagggctcaaagaatg 2562 2581 SEQ ID NO: 1055 cattcctttaaccattcct9491 9510 1 6 SEQ ID NO: 53 gaagggctcaaagaatgac 2564 2583 SEQ ID NO:1056 gtcagtcttcaggctcttc 7914 7933 1 6 SEQ ID NO: 54 caaagaatgacttttttct2572 2591 SEQ ID NO: 1057 agaaggatggcattttttg 14000 14019 1 6 SEQ ID NO:55 catggagaatgcctttgaa 2603 2622 SEQ ID NO: 1058 ttcagagccaaagtccatg7119 7138 1 6 SEQ ID NO: 56 ggagccaaggctggagtaa 2679 2698 SEQ ID NO:1059 ttactccaacgccagctcc 3050 3069 1 6 SEQ ID NO: 57 tcattccttccccaaagag2884 2903 SEQ ID NO: 1060 ctctctggggcatctatga 5139 5158 1 6 SEQ ID NO:58 acctatgagctccagagag 3165 3184 SEQ ID NO: 1061 ctctcaagaccacagaggt12976 12995 1 6 SEQ ID NO: 59 gggcaaaacgtcttacaga 3365 3384 SEQ ID NO:1062 tctgaaagacaacgtgccc 12317 12336 1 6 SEQ ID NO: 60accctggacattcagaaca 3387 3406 SEQ ID NO: 1063 tgttgctaaggttcagggt 56755694 1 6 SEQ ID NO: 61 atgggcgacctaagttgtg 3429 3448 SEQ ID NO: 1064cacaaattagtttcaccat 8941 8960 1 6 SEQ ID NO: 62 gatgaagagaagattgaat 36183637 SEQ ID NO: 1065 attccagcttccccacatc 8330 8349 1 6 SEQ ID NO: 63caatgtagataccaaaaaa 3656 3675 SEQ ID NO: 1066 ttttttggaaatgccattg 86438662 1 6 SEQ ID NO: 64 gtagataccaaaaaaatga 3660 3679 SEQ ID NO: 1067tcatgtgatgggtctctac 4371 4390 1 6 SEQ ID NO: 65 gcttcagttcatttggact 45094528 SEQ ID NO: 1068 agtcaagaaggacttaagc 5304 5323 1 6 SEQ ID NO: 66tttgtttgtcaaagaagtc 4544 4563 SEQ ID NO: 1069 gacttcagagaaatacaaa 1140011419 1 6 SEQ ID NO: 67 ttgtttgtcaaagaagtca 4545 4564 SEQ ID NO: 1070tgacttcagagaaatacaa 11399 11418 1 6 SEQ ID NO: 68 tggcaatgggaaactcgct5846 5865 SEQ ID NO: 1071 agcgagaatcaccctgcca 8219 8238 1 6 SEQ ID NO:69 aacctctggcatttacttt 5917 5936 SEQ ID NO: 1072 aaaggagatgtcaagggtt10599 10618 1 6 SEQ ID NO: 70 catttactttctctcatga 5926 5945 SEQ ID NO:1073 tcatttgaaagaataaatg 7026 7045 1 6 SEQ ID NO: 71 aaagtcagtgccctgctta6009 6028 SEQ ID NO: 1074 taagaaccttactgacttt 7784 7803 1 6 SEQ ID NO:72 tcccattttttgagacctt 6322 6341 SEQ ID NO: 1075 aaggacttcaggaatggga12004 12023 1 6 SEQ ID NO: 73 catcaatattgatcaattt 6413 6432 SEQ ID NO:1076 aaattaaaaagtcttgatg 6732 6751 1 6 SEQ ID NO: 74 taaagatagttatgattta6665 6684 SEQ ID NO: 1077 taaaccaaaacttggttta 9019 9038 1 6 SEQ ID NO:75 tattgatgaaatcattgaa 6713 6732 SEQ ID NO: 1078 ttcaaagacttaaaaaata8007 8026 1 6 SEQ ID NO: 76 atgatctacatttgtttat 6790 6809 SEQ ID NO:1079 ataaagaaattaaagtcat 7380 7399 1 6 SEQ ID NO: 77 agagacacatacagaatat6919 6938 SEQ ID NO: 1080 atatattgtcagtgcctct 13382 13401 1 6 SEQ ID NO:78 gacacatacagaatataga 6922 6941 SEQ ID NO: 1081 tctaaattcagttcttgtc11327 11346 1 6 SEQ ID NO: 79 agcatgtcaaacactttgt 7054 7073 SEQ ID NO:1082 acaaagtcagtgccctgct 6007 6026 1 6 SEQ ID NO: 80 tttttagaggaaaccaagg7515 7534 SEQ ID NO: 1083 cctttgtgtacaccaaaaa 11230 11249 1 6 SEQ ID NO:81 ttttagaggaaaccaaggc 7516 7535 SEQ ID NO: 1084 gcctttgtgtacaccaaaa11229 11248 1 6 SEQ ID NO: 82 ggaagatagacttcctgaa 9307 9326 SEQ ID NO:1085 ttcagaaatactgttttcc 12824 12843 1 6 SEQ ID NO: 83cactgtttctgagtcccag 9334 9353 SEQ ID NO: 1086 ctgggacctaccaagagtg 1252312542 1 6 SEQ ID NO: 84 cacaaatcctttggctgtg 9668 9687 SEQ ID NO: 1087cacatttcaaggaattgtg 10063 10082 1 6 SEQ ID NO: 85 ttcctggatacactgttcc9853 9872 SEQ ID NO: 1088 ggaactgttgactcaggaa 12569 12588 1 6 SEQ ID NO:86 gaaatctcaagctttctct 10042 10061 SEQ ID NO: 1089 agagccaggtcgagctttc11044 11063 1 6 SEQ ID NO: 87 tttcttcatcttcatctgt 10210 10229 SEQ ID NO:1090 acagctgaaagagatgaaa 13055 13074 1 6 SEQ ID NO: 88tctaccgctaaaggagcag 10521 10540 SEQ ID NO: 1091 ctgcacgctttgaggtaga11761 11780 1 6 SEQ ID NO: 89 ctaccgctaaaggagcagt 10522 10541 SEQ ID NO:1092 actgcacgctttgaggtag 11760 11779 1 6 SEQ ID NO: 90agggcctctttttcaccaa 10831 10850 SEQ ID NO: 1093 ttggccaggaagtggccct10957 10976 1 6 SEQ ID NO: 91 ttctccatccctgtaaaag 11265 11284 SEQ ID NO:1094 ctttttcaccaacggagaa 10838 10857 1 6 SEQ ID NO: 92gaaaaacaaagcagattat 11816 11835 SEQ ID NO: 1095 ataaactgcaagatttttc13600 13619 1 6 SEQ ID NO: 93 actcactcattgattttct 12682 12701 SEQ ID NO:1096 agaaaatcaggatctgagt 14027 14046 1 6 SEQ ID NO: 94taaactaatagatgtaatc 12890 12909 SEQ ID NO: 1097 gattaccaccagcagttta13578 13597 1 6 SEQ ID NO: 95 caaaacgagcttcaggaag 13200 13219 SEQ ID NO:1098 cttcgtgaagaatattttg 13260 13279 1 6 SEQ ID NO: 96tggaataatgctcagtgtt 2366 2385 SEQ ID NO: 1099 aacacttacttgaattcca 1066210681 3 5 SEQ ID NO: 97 gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1100cttcagagaaatacaaatc 11402 11421 3 5 SEQ ID NO: 98 atttgaaatccaaagaagt2401 2420 SEQ ID NO: 1101 acttcagagaaatacaaat 11401 11420 3 5 SEQ ID NO:99 atcaacagccgcttctttg 990 1009 SEQ ID NO: 1102 caaagaagtcaagattgat 45534572 2 5 SEQ ID NO: 100 tgttttgaagactctccag 1082 1101 SEQ ID NO: 1103ctggaaagttaaaacaaca 6955 6974 2 5 SEQ ID NO: 101 cccttctgatagatgtggt1324 1343 SEQ ID NO: 1104 accaaagctggcaccaggg 13961 13980 2 5 SEQ ID NO:102 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO: 1105 aaagccattcagtctctca12963 12982 2 5 SEQ ID NO: 103 atttgaaatccaaagaagt 2401 2420 SEQ ID NO:1106 acttttctaaacttgaaat 9055 9074 2 5 SEQ ID NO: 104atccaaagaagtcccggaa 2408 2427 SEQ ID NO: 1107 ttccggggaaacctgggat 1272112740 2 5 SEQ ID NO: 105 agagcctacctccgcatct 2430 2449 SEQ ID NO: 1108agatggtacgttagcctct 11921 11940 2 5 SEQ ID NO: 106 aatgcctttgaactcccca2610 2629 SEQ ID NO: 1109 tgggaactacaatttcatt 7012 7031 2 5 SEQ ID NO:107 gaagtccaaattccggatt 3297 3316 SEQ ID NO: 1110 aatcttcaatttattcttc13815 13834 2 5 SEQ ID NO: 108 tgcaagcagaagccagaag 3496 3515 SEQ ID NO:1111 cttcaggttccatcgtgca 11376 11395 2 5 SEQ ID NO: 109gaagagaagattgaatttg 3621 3640 SEQ ID NO: 1112 caaaacctactgtctcttc 1045910478 2 5 SEQ ID NO: 110 atgctaaaggcacatatgg 4597 4816 SEQ ID NO: 1113ccatatgaaagtcaagcat 12656 12675 2 5 SEQ ID NO: 111 tccctcacctccacctctg4737 4756 SEQ ID NO: 1114 cagattctcagatgaggga 8912 8931 2 5 SEQ ID NO:112 atttacagctctgacaagt 5427 5446 SEQ ID NO: 1115 acttttctaaacttgaaat9055 9074 2 5 SEQ ID NO: 113 aggagcctaccaaaataat 5594 5613 SEQ ID NO:1116 attatgttgaaacagtcct 11830 11849 2 5 SEQ ID NO: 114aaagctgaagcacatcaat 6401 6420 SEQ ID NO: 1117 attgttgctcatctccttt 1019410213 2 5 SEQ ID NO: 115 ctgctggaaacaacgagaa 9418 9437 SEQ ID NO: 1118ttctgattaccaccagcag 13574 13593 2 5 SEQ ID NO: 116 ttgaaggaattcttgaaaa9582 9601 SEQ ID NO: 1119 ttttaaaagaaatcttcaa 13805 13824 2 5 SEQ ID NO:117 gaagtaaaagaaaattttg 10743 10762 SEQ ID NO: 1120 caaaacctactgtctcttc10459 10478 2 5 SEQ ID NO: 118 tgaagaagatggcaaattt 11984 12003 SEQ IDNO: 1121 aaatgtcagctcttgttca 10894 10913 2 5 SEQ ID NO: 119aggatctgagttattttgc 14035 14054 SEQ ID NO: 1122 gcaagtcagcccagttcct10920 10939 2 5 SEQ ID NO: 120 gtgcccttctcggttgctg 18 37 SEQ ID NO: 1123cagccattgacatgagcac 5740 5759 1 5 SEQ ID NO: 121 ggcgctgcctgcgctgctg 146165 SEQ ID NO: 1124 cagctccacagactccgcc 3062 3081 1 5 SEQ ID NO: 122ctgcgctgctgctgctgct 154 173 SEQ ID NO: 1125 agcagaaggtgcgaagcag 32243243 1 5 SEQ ID NO: 123 gctgctggcgggcgccagg 170 189 SEQ ID NO: 1126cctggattccacatgcagc 11846 11865 1 5 SEQ ID NO: 124 aagaggaaatgctggaaaa193 212 SEQ ID NO: 1127 tttttcttcactacatctt 2584 2603 1 5 SEQ ID NO: 125ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1128 ccagacttccacatcccag 39153934 1 5 SEQ ID NO: 126 tggagtccctgggactgct 296 315 SEQ ID NO: 1129agcatgcctagtttctcca 9945 9964 1 5 SEQ ID NO: 127 ggagtccctgggactgctg 297316 SEQ ID NO: 1130 cagcatgcctagtttctcc 9944 9963 1 5 SEQ ID NO: 128tgggactgctgattcaaga 305 324 SEQ ID NO: 1131 tcttccatcacttgaccca 20422061 1 5 SEQ ID NO: 129 ctgctgattcaagaagtgc 310 329 SEQ ID NO: 1132gcacaccttgacattgcag 11079 11098 1 5 SEQ ID NO: 130 tgccaccaggatcaactgc326 345 SEQ ID NO: 1133 gcaggctgaactggtggca 2717 2736 1 5 SEQ ID NO: 131gccaccaggatcaactgca 327 346 SEQ ID NO: 1134 tgcaggctgaactggtggc 27162735 1 5 SEQ ID NO: 132 tgcaaggttgagctggagg 342 361 SEQ ID NO: 1135cctccacctctgatctgca 4744 4763 1 5 SEQ ID NO: 133 caaggttgagctggaggtt 344363 SEQ ID NO: 1136 aacccctacatgaagcttg 13755 13774 1 5 SEQ ID NO: 134ctctgcagcttcatcctga 369 388 SEQ ID NO: 1137 tcaggaagcttctcaagag 1321113230 1 5 SEQ ID NO: 135 cagcttcatcctgaagacc 374 393 SEQ ID NO: 1138ggtcttgagttaaatgctg 4977 4996 1 5 SEQ ID NO: 136 gcttcatcctgaagaccag 376395 SEQ ID NO: 1139 ctggacgctaagaggaagc 855 874 1 5 SEQ ID NO: 137tcatcctgaagaccagcca 379 398 SEQ ID NO: 1140 tggcatggcattatgatga 36043623 1 5 SEQ ID NO: 138 gaaaaccaagaactctgag 452 471 SEQ ID NO: 1141ctcaaccttaatgattttc 8286 8305 1 5 SEQ ID NO: 139 agaactctgaggagtttgc 460479 SEQ ID NO: 1142 gcaagctatacagtattct 8377 8396 1 5 SEQ ID NO: 140tctgaggagtttgctgcag 465 484 SEQ ID NO: 1143 ctgcaggggatcccccaga 25262545 1 5 SEQ ID NO: 141 tttgctgcagccatgtcca 474 493 SEQ ID NO: 1144tggaagtgtcagtggcaaa 10372 10391 1 5 SEQ ID NO: 142 caagaggggcatcatttct578 597 SEQ ID NO: 1145 agaataaatgacgttcttg 7035 7054 1 5 SEQ ID NO: 143tcactttaccgtcaagacg 674 693 SEQ ID NO: 1146 cgtctacactatcatgtga 43604379 1 5 SEQ ID NO: 144 tttaccgtcaagacgagga 678 697 SEQ ID NO: 1147tccttgacatgttgataaa 7366 7385 1 5 SEQ ID NO: 145 cactggacgctaagaggaa 853872 SEQ ID NO: 1148 ttccagaaagcagccagtg 12498 12517 1 5 SEQ ID NO: 146aggaagcatgtggcagaag 867 886 SEQ ID NO: 1149 cttcatacacattaatcct 998810007 1 5 SEQ ID NO: 147 caaggagcaacacctcttc 893 912 SEQ ID NO: 1150gaagtagtactgcatcttg 6835 6854 1 5 SEQ ID NO: 148 acagactttgaaacttgaa 959978 SEQ ID NO: 1151 ttcaattcttcaatgctgt 10500 10519 1 5 SEQ ID NO: 149tgatgaagcagtcacatct 1187 1206 SEQ ID NO: 1152 agatttgaggattccatca 79767995 1 5 SEQ ID NO: 150 agcagtcacatctctcttg 1193 1212 SEQ ID NO: 1153caaggagaaactgactgct 6524 6543 1 5 SEQ ID NO: 151 ccagccccatcactttaca1231 1250 SEQ ID NO: 1154 tgtagtctcctggtgctgg 5094 5113 1 5 SEQ ID NO:152 ctccactcacatcctccag 1280 1299 SEQ ID NO: 1155 ctggagcttagtaatggag8709 8728 1 5 SEQ ID NO: 153 catgccaacccccttctga 1314 1333 SEQ ID NO:1156 tcagatgagggaacacatg 8919 8938 1 5 SEQ ID NO: 154gagagatcttcaacatggc 1390 1409 SEQ ID NO: 1157 gccaccctggaactctctc 1086910888 1 5 SEQ ID NO: 155 tcaacatggcgagggatca 1399 1418 SEQ ID NO: 1158tgatcccacctctcattga 2965 2984 1 5 SEQ ID NO: 156 ccaccttgtatgcgctgag1429 1448 SEQ ID NO: 1159 ctcagggatctgaaggtgg 8187 8206 1 5 SEQ ID NO:157 gtcaacaactatcataaga 1455 1474 SEQ ID NO: 1160 tcttgagttaaatgctgac4979 4998 1 5 SEQ ID NO: 158 tggacattgctaattacct 1501 1520 SEQ ID NO:1161 aggtatattcgaaagtcca 12799 12818 1 5 SEQ ID NO: 159ggacattgctaattacctg 1502 1521 SEQ ID NO: 1162 caggtatattcgaaagtcc 1279812817 1 5 SEQ ID NO: 160 ttctgcgggtcattggaaa 1573 1592 SEQ ID NO: 1163tttcacatgccaaggagaa 6514 6533 1 5 SEQ ID NO: 161 ccagaactcaagtcttcaa1620 1639 SEQ ID NO: 1164 ttgaagtgtagtctcctgg 5088 5107 1 5 SEQ ID NO:162 agtcttcaatcctgaaatg 1630 1649 SEQ ID NO: 1165 catttctgattggtggact7757 7776 1 5 SEQ ID NO: 163 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO:1166 aaagtgccacttttactca 6183 6202 1 5 SEQ ID NO: 164agcaagtgaagaactttgt 1870 1889 SEQ ID NO: 1167 acaaagtcagtgccctgct 60076026 1 5 SEQ ID NO: 165 tctgaaagaatctcaactt 1964 1983 SEQ ID NO: 1168aagtccataatggttcaga 12811 12830 1 5 SEQ ID NO: 166 actgtcatggacttcagaa1986 2005 SEQ ID NO: 1169 ttctgaatatattgtcagt 13376 13395 1 5 SEQ ID NO:167 acttgacccagcctcagcc 2051 2070 SEQ ID NO: 1170 ggctcaccctgagagaagt12391 12410 1 5 SEQ ID NO: 168 tccaaataactaccttcct 2096 2115 SEQ ID NO:1171 aggaagatatgaagatgga 4712 4731 1 5 SEQ ID NO: 169actaccctcactgcctttg 2133 2152 SEQ ID NO: 1172 caaatttgtggagggtagt 1031910338 1 5 SEQ ID NO: 170 ttggatttgcttcagctga 2149 2168 SEQ ID NO: 1173tcagtataagtacaaccaa 9392 9411 1 5 SEQ ID NO: 171 ttggaagctctttttggga2211 2230 SEQ ID NO: 1174 tcccgattcacgcttccaa 11577 11596 1 5 SEQ ID NO:172 ggaagctctttttgggaag 2213 2232 SEQ ID NO: 1175 cttcagaaagctaccttcc7929 7948 1 5 SEQ ID NO: 173 tttttcccagacagtgtca 2238 2257 SEQ ID NO:1176 tgaccttctctaagcaaaa 4876 4895 1 5 SEQ ID NO: 174agacagtgtcaacaaagct 2246 2265 SEQ ID NO: 1177 agcttggttttgccagtct 24582477 1 5 SEQ ID NO: 175 ctttggctataccaaagat 2321 2340 SEQ ID NO: 1178atctcgtgtctaggaaaag 5968 5987 1 5 SEQ ID NO: 176 caaagatgataaacatgag2333 2352 SEQ ID NO: 1179 ctcaaggataacgtgtttg 12609 12628 1 5 SEQ ID NO:177 gatatggtaaatggaataa 2355 2374 SEQ ID NO: 1180 ttatcttattaattatatc13079 13098 1 5 SEQ ID NO: 178 ggaataatgctcagtgttg 2367 2386 SEQ ID NO:1181 caacacttacttgaattcc 10661 10680 1 5 SEQ ID NO: 179tttgaaatccaaagaagtc 2402 2421 SEQ ID NO: 1182 gacttcagagaaatacaaa 1140011419 1 5 SEQ ID NO: 180 gatcccccagatgattgga 2534 2553 SEQ ID NO: 1183tccaatttccctgtggatc 3681 3700 1 5 SEQ ID NO: 181 cagatgattggagaggtca2541 2560 SEQ ID NO: 1184 tgaccacacaaacagtctg 5363 5382 1 5 SEQ ID NO:182 agaatgacttttttcttca 2575 2594 SEQ ID NO: 1185 tgaagtccggattcattct11015 11034 1 5 SEQ ID NO: 183 gaactccccactggagctg 2619 2638 SEQ ID NO:1186 cagctcaaccgtacagttc 11861 11880 1 5 SEQ ID NO: 184atatcttcatctggagtca 2652 2671 SEQ ID NO: 1187 tgacttcagtgcagaatat 1196611985 1 5 SEQ ID NO: 185 gtcattgctcccggagcca 2667 2886 SEQ ID NO: 1188tggccccgtttaccatgac 5809 5828 1 5 SEQ ID NO: 186 gctgaagtttatcattcct2873 2892 SEQ ID NO: 1189 aggaggctttaagttcagc 7600 7619 1 5 SEQ ID NO:187 attccttccccaaagagac 2886 2905 SEQ ID NO: 1190 gtctcttcctccatggaat10470 10489 1 5 SEQ ID NO: 188 ctcattgagaacaggcagt 2976 2995 SEQ ID NO:1191 actgactgcacgctttgag 11756 11775 1 5 SEQ ID NO: 189ttgagcagtattctgtcag 3142 3161 SEQ ID NO: 1192 ctgagagaagtgtcttcaa 1239912418 1 5 SEQ ID NO: 190 accttgtccagtgaagtcc 3285 3304 SEQ ID NO: 1193ggacggtactgtcccaggt 12784 12803 1 5 SEQ ID NO: 191 ccagtgaagtccaaattcc3292 3311 SEQ ID NO: 1194 ggaaggcagagtttactgg 9148 9167 1 5 SEQ ID NO:192 acattcagaacaagaaaat 3394 3413 SEQ ID NO: 1195 atttcctaaagctggatgt11167 11186 1 5 SEQ ID NO: 193 gaaaaatcaagggtgttat 3463 3482 SEQ ID NO:1196 ataaactgcaagatttttc 13600 13619 1 5 SEQ ID NO: 194aaatcaagggtgttatttc 3466 3485 SEQ ID NO: 1197 gaaacaatgcattagattt 97459764 1 5 SEQ ID NO: 195 tggcattatgatgaagaga 3609 3628 SEQ ID NO: 1198tctcccgtgtataatgcca 11781 11800 1 5 SEQ ID NO: 196 aagagaagattgaatttga3622 3641 SEQ ID NO: 1199 tcaaaacctactgtctctt 10458 10477 1 5 SEQ ID NO:197 aaatgacttccaatttccc 3673 3692 SEQ ID NO: 1200 gggaactacaatttcattt7013 7032 1 5 SEQ ID NO: 198 atgacttccaatttccctg 3675 3694 SEQ ID NO:1201 caggctgattacgagtcat 4917 4936 1 5 SEQ ID NO: 199acttccaatttccctgtgg 3678 3697 SEQ ID NO: 1202 ccacgaaaaatatggaagt 1036010379 1 5 SEQ ID NO: 200 agttgcaatgagctcatgg 3803 3822 SEQ ID NO: 1203ccatcagttcagataaact 7989 8008 1 5 SEQ ID NO: 201 tttgcaagaccacctcaat3860 3879 SEQ ID NO: 1204 attgacctgtccattcaaa 13671 13690 1 5 SEQ ID NO:202 gaaggagttcaacctccag 3884 3903 SEQ ID NO: 1205 ctggaattgtcattccttc11728 11747 1 5 SEQ ID NO: 203 acttccacatcccagaaaa 3919 3938 SEQ ID NO:1206 ttttaacaaaagtggaagt 6821 6840 1 5 SEQ ID NO: 204ctcttcttaaaaagcgatg 3939 3958 SEQ ID NO: 1207 catcactgccaaaggagag 84868505 1 5 SEQ ID NO: 205 aaaagcgatggccgggtca 3948 3967 SEQ ID NO: 1208tgactcactcattgatttt 12680 12699 1 5 SEQ ID NO: 206 ttcctttgccttttggtgg4003 4022 SEQ ID NO: 1209 ccacaaacaatgaagggaa 9256 9275 1 5 SEQ ID NO:207 caagtctgtgggattccat 4079 4098 SEQ ID NO: 1210 atgggaaaaaacaggcttg9566 9585 1 5 SEQ ID NO: 208 aagtccctacttttaccat 4117 4136 SEQ ID NO:1211 atgggaagtataagaactt 4834 4853 1 5 SEQ ID NO: 209tgcctctcctgggtgttct 4159 4178 SEQ ID NO: 1212 agaaaaacaaacacaggca 96439662 1 5 SEQ ID NO: 210 accagcacagaccatttca 4242 4261 SEQ ID NO: 1213tgaagtgtagtctcctggt 5089 5108 1 5 SEQ ID NO: 211 ccagcacagaccatttcag4243 4262 SEQ ID NO: 1214 ctgaaatacaatgctctgg 5511 5530 1 5 SEQ ID NO:212 actatcatgtgatgggtct 4367 4386 SEQ ID NO: 1215 agacacctgattttatagt7948 7967 1 5 SEQ ID NO: 213 accacagatgtctgcttca 4496 4515 SEQ ID NO:1216 tgaaggctgactctgtggt 4282 4301 1 5 SEQ ID NO: 214ccacagatgtctgcttcag 4497 4516 SEQ ID NO: 1217 ctgagcaacaaatttgtgg 1031110330 1 5 SEQ ID NO: 215 tttggactccaaaaagaaa 4520 4539 SEQ ID NO: 1218tttctctcatgattacaaa 5933 5952 1 5 SEQ ID NO: 216 tcaaagaagtcaagattga4552 4571 SEQ ID NO: 1219 tcaaggataacgtgtttga 12610 12629 1 5 SEQ ID NO:217 atgagaactacgagctgac 4798 4817 SEQ ID NO: 1220 gtcagatattgttgctcat10187 10206 1 5 SEQ ID NO: 218 ttaaaatctgacaccaatg 4818 4837 SEQ ID NO:1221 cattcattgaagatgttaa 7342 7361 1 5 SEQ ID NO: 219gaagtataagaactttgcc 4838 4857 SEQ ID NO: 1222 ggcaaatttgaaggacttc 1199412013 1 5 SEQ ID NO: 220 aagtataagaactttgcca 4839 4858 SEQ ID NO: 1223tggcaaatttgaaggactt 11993 12012 1 5 SEQ ID NO: 221 ttcttcagcctgctttctg4941 4960 SEQ ID NO: 1224 cagaatccagatacaagaa 6884 6903 1 5 SEQ ID NO:222 ctggatcactaaattccca 4957 4976 SEQ ID NO: 1225 tgggtctttccagagccag11033 11052 1 5 SEQ ID NO: 223 aaattaatagtggtgctca 5014 5033 SEQ ID NO:1226 tgagaagccccaagaattt 6248 6267 1 5 SEQ ID NO: 224agtgcaacgaccaacttga 5073 5092 SEQ ID NO: 1227 tcaaattcctggatacact 98489867 1 5 SEQ ID NO: 225 ctgggaagtgcttatcagg 5238 5257 SEQ ID NO: 1228cctgaccttcacataccag 8310 8329 1 5 SEQ ID NO: 226 gcaaaaacattttcaactt5278 5297 SEQ ID NO: 1229 aagtaaaagaaaattttgc 10744 10763 1 5 SEQ ID NO:227 aaaaacattttcaacttca 5280 5299 SEQ ID NO: 1230 tgaagtaaaagaaaatttt10742 10761 1 5 SEQ ID NO: 228 tcagtcaagaaggacttaa 5302 5321 SEQ ID NO:1231 ttaaggacttccattctga 13363 13382 1 5 SEQ ID NO: 229tcaaatgacatgatgggct 5325 5344 SEQ ID NO: 1232 agcccatcaatatcattga 62056224 1 5 SEQ ID NO: 230 cacacaaacagtctgaaca 5367 5386 SEQ ID NO: 1233tgtttcaactgcctttgtg 11219 11238 1 5 SEQ ID NO: 231 tcttcaaaacttgacaaca5409 5428 SEQ ID NO: 1234 tgttttcctatttccaaga 12835 12854 1 5 SEQ ID NO:232 caagttttataagcaaact 5441 5460 SEQ ID NO: 1235 agttattttgctaaacttg14043 14062 1 5 SEQ ID NO: 233 tggtaactactttaaacag 5488 5507 SEQ ID NO:1236 ctgtttttagaggaaacca 7512 7531 1 5 SEQ ID NO: 234aacagtgacctgaaataca 5502 5521 SEQ ID NO: 1237 tgtatagcaaattcctgtt 58905909 1 5 SEQ ID NO: 235 gggaaactacggctagaac 5544 5563 SEQ ID NO: 1238gttccttccatgatttccc 10933 10952 1 5 SEQ ID NO: 236 aacacatctatgccatctc5620 5639 SEQ ID NO: 1239 gagacagcatcttcgtgtt 11204 11223 1 5 SEQ ID NO:237 tcagcaagctataaagcag 5652 5671 SEQ ID NO: 1240 ctgctaagaaccttactga7780 7799 1 5 SEQ ID NO: 238 gcagacactgttgctaagg 5667 5686 SEQ ID NO:1241 cctttcaagcactgactgc 11746 11765 1 5 SEQ ID NO: 239tctggggagaacatactgg 5866 5885 SEQ ID NO: 1242 ccaggttttccacaccaga 80388057 1 5 SEQ ID NO: 240 ttctctcatgattacaaag 5934 5953 SEQ ID NO: 1243ctttttcaccaacggagaa 10838 10857 1 5 SEQ ID NO: 241 ctgagcagacaggcacctg6034 6053 SEQ ID NO: 1244 caggaggctttaagttcag 7599 7618 1 5 SEQ ID NO:242 caatttaacaacaatgaat 6066 6085 SEQ ID NO: 1245 attccttcctttacaattg8082 8101 1 5 SEQ ID NO: 243 tggacgaactctggctgac 6140 6159 SEQ ID NO:1246 gtcagcccagttccttcca 10924 10943 1 5 SEQ ID NO: 244cttttactcagtgagccca 6192 6211 SEQ ID NO: 1247 tgggctaaacgtatgaaag 78277846 1 5 SEQ ID NO: 245 tcattgatgctttagagat 6217 6236 SEQ ID NO: 1248atcttcataagttcaatga 13174 13193 1 5 SEQ ID NO: 246 aaaaccaagatgttcactc6295 6314 SEQ ID NO: 1249 gagtgaaatgctgtttttt 8630 8649 1 5 SEQ ID NO:247 aggaatcgacaaaccatta 6357 6376 SEQ ID NO: 1250 taatgattttcaagttcct8294 8313 1 5 SEQ ID NO: 248 tagttgtactggaaaacgt 6376 6395 SEQ ID NO:1251 acgttagcctctaagacta 11928 11947 1 5 SEQ ID NO: 249ggaaaacgtacagagaaag 6386 6405 SEQ ID NO: 1252 cttttacaattcattttcc 1301413033 1 5 SEQ ID NO: 250 gaaaacgtacagagaaagc 6387 6406 SEQ ID NO: 1253gctttctcttccacatttc 10052 10071 1 5 SEQ ID NO: 251 aaagctgaagcacatcaat6401 6420 SEQ ID NO: 1254 attgatgttagagtgcttt 6984 7003 1 5 SEQ ID NO:252 aagctgaagcacatcaata 6402 6421 SEQ ID NO: 1255 tattgatgttagagtgctt6983 7002 1 5 SEQ ID NO: 253 tgaagcacatcaatattga 6406 6425 SEQ ID NO:1256 tcaaccttaatgattttca 8287 8306 1 5 SEQ ID NO: 254atcaatattgatcaatttg 6414 6433 SEQ ID NO: 1257 caaagccatcactgatgat 16601679 1 5 SEQ ID NO: 255 taatgattatctgaattca 6476 6495 SEQ ID NO: 1258tgaaatcattgaaaaatta 6719 6738 1 5 SEQ ID NO: 256 gattatctgaattcattca6480 6499 SEQ ID NO: 1259 tgaagtagctgagaaaatc 7094 7113 1 5 SEQ ID NO:257 aattgggagagacaagttt 6498 6517 SEQ ID NO: 1260 aaacattcctttaacaatt9488 9507 1 5 SEQ ID NO: 258 aaaatagctattgctaata 6693 6712 SEQ ID NO:1261 tattgaaaatattgatttt 6806 6825 1 5 SEQ ID NO: 259aaaattaaaaagtcttgat 6731 6750 SEQ ID NO: 1262 atcatatccgtgtaatttt 67576776 1 5 SEQ ID NO: 260 ttgaaaatattgattttaa 6808 6827 SEQ ID NO: 1263ttaatcttcataagttcaa 13171 13190 1 5 SEQ ID NO: 261 agacatccagcacctagct6938 6957 SEQ ID NO: 1264 agcttggttttgccagtct 2458 2477 1 5 SEQ ID NO:262 caatttcatttgaaagaat 7021 7040 SEQ ID NO: 1265 attccttcctttacaattg8082 8101 1 5 SEQ ID NO: 263 aggttttaatggataaatt 7174 7193 SEQ ID NO:1266 aattgttgaaagaaaacct 13147 13166 1 5 SEQ ID NO: 264cagaagctaagcaatgtcc 7233 7252 SEQ ID NO: 1267 ggacaaggcccagaatctg 1254512564 1 5 SEQ ID NO: 265 taagataaaagattacttt 7262 7281 SEQ ID NO: 1268aaagaaaacctatgcctta 13155 13174 1 5 SEQ ID NO: 266 aaagattactttgagaaat7269 7288 SEQ ID NO: 1269 atttcttaaacattccttt 9481 9500 1 5 SEQ ID NO:267 gagaaattagttggattta 7281 7300 SEQ ID NO: 1270 taaagccattcagtctctc12962 12981 1 5 SEQ ID NO: 268 atttattgatgatgctgtc 7295 7314 SEQ ID NO:1271 gacatgttgataaagaaat 7371 7390 1 5 SEQ ID NO: 269gaattatcttttaaaacat 7326 7345 SEQ ID NO: 1272 atgtatcaaatggacattc 76777696 1 5 SEQ ID NO: 270 ttaccaccagtttgtagat 7403 7422 SEQ ID NO: 1273atctggaaccttgaagtaa 10731 10750 1 5 SEQ ID NO: 271 ttgcagtgtatctggaaag7540 7559 SEQ ID NO: 1274 cttttcacattagatgcaa 8412 8431 1 5 SEQ ID NO:272 cattcagcaggaacttcaa 7691 7710 SEQ ID NO: 1275 ttgaaggacttcaggaatg12001 12020 1 5 SEQ ID NO: 273 acacctgattttatagtcc 7950 7969 SEQ ID NO:1276 ggactcaaggataacgtgt 12606 12625 1 5 SEQ ID NO: 274ggattccatcagttcagat 7984 8003 SEQ ID NO: 1277 atcttcaatgattatatcc 1311613135 1 5 SEQ ID NO: 275 ttgtagaaatgaaagtaaa 8104 8123 SEQ ID NO: 1278tttatgattatgtcaacaa 12352 12371 1 5 SEQ ID NO: 276 ctgaacagtgagctgcagt8148 8167 SEQ ID NO: 1279 actggacttctctagtcag 8801 8820 1 5 SEQ ID NO:277 aatccaatctcctcttttc 8399 8418 SEQ ID NO: 1280 gaaaaatgaagtccggatt11009 11028 1 5 SEQ ID NO: 278 attttgattttcaagcaaa 8524 8543 SEQ ID NO:1281 tttgcaagttaaagaaaat 14015 14034 1 5 SEQ ID NO: 279ttttgattttcaagcaaat 8525 8544 SEQ ID NO: 1282 atttgatttaagtgtaaaa 96149633 1 5 SEQ ID NO: 280 tgattttcaagcaaatgca 8528 8547 SEQ ID NO: 1283tgcaagttaaagaaaatca 14017 14036 1 5 SEQ ID NO: 281 atgctgttttttggaaatg8637 8656 SEQ ID NO: 1284 cattggtaggagacagcat 11195 11214 1 5 SEQ ID NO:282 tgctgttttttggaaatgc 8638 8657 SEQ ID NO: 1285 gcattggtaggagacagca11194 11213 1 5 SEQ ID NO: 283 aaaaaaatacactggagct 8698 8717 SEQ ID NO:1286 agctagagggcctcttttt 10825 10844 1 5 SEQ ID NO: 284actggagcttagtaatgga 8708 8727 SEQ ID NO: 1287 tccactcacatcctccagt 12811300 1 5 SEQ ID NO: 285 cttctggaaaagggtcatg 8878 8897 SEQ ID NO: 1288catgaacccctacatgaag 13751 13770 1 5 SEQ ID NO: 286 ggaaaagggtcatggaaat8883 8902 SEQ ID NO: 1289 atttgaaagttcgttttcc 9274 9293 1 5 SEQ ID NO:287 gggcctgccccagattctc 8902 8921 SEQ ID NO: 1290 gagaacattatggaggccc9432 9451 1 5 SEQ ID NO: 288 ttctcagatgagggaacac 8916 8935 SEQ ID NO:1291 gtgtcttcaaagctgagaa 12408 12427 1 5 SEQ ID NO: 289gatgagggaacacatgaat 8922 8941 SEQ ID NO: 1292 attccagcttccccacatc 83308349 1 5 SEQ ID NO: 290 ctttggactgtccaataag 8978 8997 SEQ ID NO: 1293cttatgggatttcctaaag 11159 11178 1 5 SEQ ID NO: 291 gcatccacaaacaatgaag9252 9271 SEQ ID NO: 1294 cttcatctgtcattgatgc 10219 10238 1 5 SEQ ID NO:292 cacaaacaatgaagggaat 9257 9276 SEQ ID NO: 1295 attccctgaagttgatgtg11480 11499 1 5 SEQ ID NO: 293 ccaaaatttctctgctgga 9407 9426 SEQ ID NO:1296 tccatcacaaatcctttgg 9663 9682 1 5 SEQ ID NO: 294caaaatttctctgctggaa 9408 9427 SEQ ID NO: 1297 ttccatcacaaatcctttg 96629681 1 5 SEQ ID NO: 295 tctgctggaaacaacgaga 9417 9436 SEQ ID NO: 1298tctcaagagttacagcaga 13221 13240 1 5 SEQ ID NO: 296 ctgctggaaacaacgagaa9418 9437 SEQ ID NO: 1299 ttctcaagagttacagcag 13220 13239 1 5 SEQ ID NO:297 agaacattatggaggccca 9433 9452 SEQ ID NO: 1300 tgggcctgccccagattct8901 8920 1 5 SEQ ID NO: 298 agaagcaaatctggatttc 9467 9486 SEQ ID NO:1301 gaaatcttcaatttattct 13813 13832 1 5 SEQ ID NO: 299tttctctctatgggaaaaa 9557 9576 SEQ ID NO: 1302 tttttgcaagttaaagaaa 1401314032 1 5 SEQ ID NO: 300 tcagagcatcaaatccttt 9704 9723 SEQ ID NO: 1303aaagaaaatcaggatctga 14025 14044 1 5 SEQ ID NO: 301 cagaaacaatgcattagat9743 9762 SEQ ID NO: 1304 atctatgccatctcttctg 5625 5644 1 5 SEQ ID NO:302 tacacattaatcctgccat 9993 10012 SEQ ID NO: 1305 atggagtctttattgtgta14081 14100 1 5 SEQ ID NO: 303 agtcagatattgttgctca 10186 10205 SEQ IDNO: 1306 tgagaactacgagctgact 4799 4818 1 5 SEQ ID NO: 304ggagggtagtcataacagt 10328 10347 SEQ ID NO: 1307 actggtggcaaaaccctcc 27262745 1 5 SEQ ID NO: 305 caaaagccgaaattccaat 10396 10415 SEQ ID NO: 1308attgaagtacctacttttg 8358 8377 1 5 SEQ ID NO: 306 aaaagccgaaattccaatt10397 10416 SEQ ID NO: 1309 aattgaagtacctactttt 8357 8376 1 5 SEQ ID NO:307 ttcaagcaagaacttaatg 10428 10447 SEQ ID NO: 1310 cattatggcccttcgtgaa13250 13269 1 5 SEQ ID NO: 308 cctcttacttttccattga 10570 10589 SEQ IDNO: 1311 tcaaaagaagcccaagagg 12939 12958 1 5 SEQ ID NO: 309tgaggccaacacttacttg 10655 10674 SEQ ID NO: 1312 caagcatctgattgactca12668 12687 1 5 SEQ ID NO: 310 cacttacttgaattccaag 10664 10683 SEQ IDNO: 1313 cttgaacacaaagtcagtg 6000 6019 1 5 SEQ ID NO: 311gaagtaaaagaaaattttg 10743 10762 SEQ ID NO: 1314 caaaaacattttcaacttc 52795298 1 5 SEQ ID NO: 312 cctggaactctctccatgg 10874 10893 SEQ ID NO: 1315ccatttacagatcttcagg 11364 11383 1 5 SEQ ID NO: 313 agctggatgtaaccaccag11176 11195 SEQ ID NO: 1316 ctggattccacatgcagct 11847 11866 1 5 SEQ IDNO: 314 aaaattccctgaagttgat 11477 11496 SEQ ID NO: 1317atcatatccgtgtaatttt 6757 6776 1 5 SEQ ID NO: 315 cagatggcattgctgcttt11605 11624 SEQ ID NO: 1318 aaagctgagaagaaatctg 12416 12435 1 5 SEQ IDNO: 316 agatggcattgctgctttg 11606 11625 SEQ ID NO: 1319caaagctgagaagaaatct 12415 12434 1 5 SEQ ID NO: 317 tgttgaaacagtcctggat11834 11853 SEQ ID NO: 1320 atccaagatgagatcaaca 13095 13114 1 5 SEQ IDNO: 318 catattcaaaactgagttg 12221 12240 SEQ ID NO: 1321caactctctgattactatg 13623 13642 1 5 SEQ ID NO: 319 aaagatttatcaaaagaag12930 12949 SEQ ID NO: 1322 cttcaatttattcttcttt 13818 13837 1 5 SEQ IDNO: 320 attttccaactaatagaag 13026 13045 SEQ ID NO: 1323cttcaaagacttaaaaaat 8006 8025 1 5 SEQ ID NO: 321 aattatatccaagatgaga13089 13108 SEQ ID NO: 1324 tctcttcctccatggaatt 10471 10490 1 5 SEQ IDNO: 322 ttcaggaagcttctcaaga 13210 13229 SEQ ID NO: 1325tcttcataagttcaatgaa 13175 13194 1 5 SEQ ID NO: 323 ttgagcaatttctgcacag13429 13448 SEQ ID NO: 1326 ctgttgaaagatttatcaa 12924 12943 1 5 SEQ IDNO: 324 ctgatatacatcacggagt 13704 13723 SEQ ID NO: 1327actcaatggtgaaattcag 7457 7476 1 5 SEQ ID NO: 325 acatcacggagttactgaa13711 13730 SEQ ID NO: 1328 ttcagaagctaagcaatgt 7231 7250 1 5 SEQ ID NO:326 actgcctatattgataaaa 13874 13893 SEQ ID NO: 1329 ttttggcaagctatacagt8372 8391 1 5 SEQ ID NO: 327 aggatggcattttttgcaa 14003 14022 SEQ ID NO:1330 ttgcaagcaagtctttcct 3005 3024 1 5 SEQ ID NO: 328ttttttgcaagttaaagaa 14012 14031 SEQ ID NO: 1331 ttctctctatgggaaaaaa 95589577 1 5 SEQ ID NO: 329 tccagaactcaagtcttca 1619 1638 SEQ ID NO: 1332tgaaatgctgttttttgga 8633 8652 3 4 SEQ ID NO: 330 agttagtgaaagaagttct1948 1967 SEQ ID NO: 1333 agaatctgtaccaggaact 12556 12575 3 4 SEQ ID NO:331 atttacagctctgacaagt 5427 5446 SEQ ID NO: 1334 acttcagagaaatacaaat11401 11420 3 4 SEQ ID NO: 332 gattatctgaattcattca 6480 6499 SEQ ID NO:1335 tgaaaccaatgacaaaatc 7421 7440 3 4 SEQ ID NO: 333gtgcccttctcggttgctg 18 37 SEQ ID NO: 1336 cagctgagcagacaggcac 6031 60502 4 SEQ ID NO: 334 attcaagcacctccggaag 245 264 SEQ ID NO: 1337cttcataagttcaatgaat 13176 13195 2 4 SEQ ID NO: 335 gactgctgattcaagaagt308 327 SEQ ID NO: 1338 acttcccaactctcaagtc 13407 13426 2 4 SEQ ID NO:336 ttgctgcagccatgtccag 475 494 SEQ ID NO: 1339 ctgggcagctgtatagcaa 58815900 2 4 SEQ ID NO: 337 agaaagatgaacctactta 547 566 SEQ ID NO: 1340taagtatgatttcaattct 10490 10509 2 4 SEQ ID NO: 338 tgaagactctccaggaact1087 1105 SEQ ID NO: 1341 agttcaatgaatttattca 13183 13202 2 4 SEQ ID NO:339 atctctcttgccacagctg 1202 1221 SEQ ID NO: 1342 cagcccagccatttgagat9229 9248 2 4 SEQ ID NO: 340 tctctcttgccacagctga 1203 1222 SEQ ID NO:1343 tcagcccagccatttgaga 9228 9247 2 4 SEQ ID NO: 341tgaggtgtccagccccatc 1223 1242 SEQ ID NO: 1344 gatgggaaagccgccctca 52085227 2 4 SEQ ID NO: 342 ccagaactcaagtcttcaa 1620 1639 SEQ ID NO: 1345ttgaaagcagaacctctgg 5907 5926 2 4 SEQ ID NO: 343 ctgaaaaagttagtgaaag1941 1960 SEQ ID NO: 1346 ctttctcgggaatattcag 10623 10642 2 4 SEQ ID NO:344 tttttcccagacagtgtca 2238 2257 SEQ ID NO: 1347 tgacaggcattttgaaaaa9722 9741 2 4 SEQ ID NO: 345 ttttcccagacagtgtcaa 2239 2258 SEQ ID NO:1348 ttgacaggcattttgaaaa 9721 9740 2 4 SEQ ID NO: 346cattcagaacaagaaaatt 3395 3414 SEQ ID NO: 1349 aattccaattttgagaatg 1040610425 2 4 SEQ ID NO: 347 tgaagagaagattgaattt 3620 3639 SEQ ID NO: 1350aaatgtcagctcttgttca 10894 10913 2 4 SEQ ID NO: 348 tttgaatggaacacaggca3636 3655 SEQ ID NO: 1351 tgccagtttgaaaaacaaa 11807 11826 2 4 SEQ ID NO:349 ttctagattcgaatatcaa 4399 4418 SEQ ID NO: 1352 ttgacatgttgataaagaa7369 7388 2 4 SEQ ID NO: 350 gattcgaatatcaaattca 4404 4423 SEQ ID NO:1353 tgaagtagaccaacaaatc 7154 7173 2 4 SEQ ID NO: 351tgcaacgaccaacttgaag 5075 5094 SEQ ID NO: 1354 cttcaggttccatcgtgca 1137611395 2 4 SEQ ID NO: 352 ttaagctctcaaatgacat 5317 5336 SEQ ID NO: 1355atgttgataaagaaattaa 7374 7393 2 4 SEQ ID NO: 353 caatttaacaacaatgaat6066 6085 SEQ ID NO: 1356 attcaaactgcctatattg 13868 13887 2 4 SEQ ID NO:354 tgaatacagccaggacttg 6080 6099 SEQ ID NO: 1357 caagagcacacggtcttca10679 10698 2 4 SEQ ID NO: 355 catcaatattgatcaattt 6413 6432 SEQ ID NO:1358 aaattccctgaagttgatg 11478 11497 2 4 SEQ ID NO: 356ttgagcatgtcaaacactt 7051 7070 SEQ ID NO: 1359 aagtaagtgctaggttcaa 93739392 2 4 SEQ ID NO: 357 tgaaggagactattcagaa 7219 7238 SEQ ID NO: 1360ttctgcacagaaatattca 13438 13457 2 4 SEQ ID NO: 358 ttcaggctcttcagaaagc7921 7940 SEQ ID NO: 1361 gcttgctaacctctctgaa 12304 12323 2 4 SEQ ID NO:359 tccacaaattgaacatccc 8779 8798 SEQ ID NO: 1362 gggacctaccaagagtgga12525 12544 2 4 SEQ ID NO: 360 tgaataccaatgctgaact 10159 10178 SEQ IDNO: 1363 agttcaatgaatttattca 13183 13202 2 4 SEQ ID NO: 361taaactaatagatgtaatc 12890 12909 SEQ ID NO: 1364 gattactatgaaaaattta13632 13651 2 4 SEQ ID NO: 362 ttgacctgtccattcaaaa 13672 13691 SEQ IDNO: 1365 ttttaaaagaaatcttcaa 13805 13824 2 4 SEQ ID NO: 363gggctgagtgcccttctcg 11 30 SEQ ID NO: 1366 cgaggccaggccgcagccc 76 95 1 4SEQ ID NO: 364 ggctgagtgcccttctcgg 12 31 SEQ ID NO: 1367ccgaggccaggccgcagcc 75 94 1 4 SEQ ID NO: 365 ctgagtgcccttctcggtt 14 33SEQ ID NO: 1368 aaccgtgcctgaatctcag 11549 11568 1 4 SEQ ID NO: 366tctcggttgctgccgctga 25 44 SEQ ID NO: 1369 tcagctgacctcatcgaga 2160 21791 4 SEQ ID NO: 367 caggccgcagcccaggagc 82 101 SEQ ID NO: 1370gctctgcagcttcatcctg 368 387 1 4 SEQ ID NO: 368 gctggcgctgcctgcgctg 143162 SEQ ID NO: 1371 cagcacagaccatttcagc 4244 4263 1 4 SEQ ID NO: 369tgctgctggcgggcgccag 169 188 SEQ ID NO: 1372 ctggatgtaaccaccagca 1117811197 1 4 SEQ ID NO: 370 ctggtctgtccaaaagatg 219 238 SEQ ID NO: 1373catcctgaagaccagccag 380 399 1 4 SEQ ID NO: 371 ctgagagttccagtggagt 283302 SEQ ID NO: 1374 actcaccctggacattcag 3383 3402 1 4 SEQ ID NO: 372tccagtggagtccctggga 291 310 SEQ ID NO: 1375 tcccggagccaaggctgga 26752694 1 4 SEQ ID NO: 373 aggttgagctggaggttcc 346 365 SEQ ID NO: 1376ggaaccctctccctcacct 4728 4747 1 4 SEQ ID NO: 374 tgagctggaggttccccag 350369 SEQ ID NO: 1377 ctgggaggcatgatgctca 9163 9182 1 4 SEQ ID NO: 375tctgcagcttcatcctgaa 370 389 SEQ ID NO: 1378 ttcaaatataatcggcaga 32613280 1 4 SEQ ID NO: 376 gccagtgcaccctgaaaga 394 413 SEQ ID NO: 1379tcttccgttctgtaatggc 5794 5813 1 4 SEQ ID NO: 377 ctctgaggagtttgctgca 464483 SEQ ID NO: 1380 tgcaagaatattttgagag 6340 6359 1 4 SEQ ID NO: 378aggtatgagctcaagctgg 492 511 SEQ ID NO: 1381 ccagtttccggggaaacct 1271612735 1 4 SEQ ID NO: 379 tcctttacccggagaaaga 535 554 SEQ ID NO: 1382tctttttgggaagcaagga 2219 2238 1 4 SEQ ID NO: 380 catcaagaggggcatcatt 575594 SEQ ID NO: 1383 aatggtcaagttcctgatg 2277 2296 1 4 SEQ ID NO: 381tcctggttcccccagagac 601 620 SEQ ID NO: 1384 gtctctgaactcagaagga 1398814007 1 4 SEQ ID NO: 382 aagaagccaagcaagtgtt 622 641 SEQ ID NO: 1385aacaaataaatggagtctt 14072 14091 1 4 SEQ ID NO: 383 aagcaagtgttgtttctgg630 649 SEQ ID NO: 1386 ccagagccaggtcgagctt 11042 11061 1 4 SEQ ID NO:384 tctggataccgtgtatgga 644 663 SEQ ID NO: 1387 tccatgtcccatttacaga11356 11375 1 4 SEQ ID NO: 385 ccactcactttaccgtcaa 670 689 SEQ ID NO:1388 ttgattttaacaaaagtgg 6817 6836 1 4 SEQ ID NO: 386aggaagggcaatgtggcaa 693 712 SEQ ID NO: 1389 ttgcaagcaagtctttcct 30053024 1 4 SEQ ID NO: 387 gcaatgtggcaacagaaat 700 719 SEQ ID NO: 1390atttccataccccgtttgc 3480 3499 1 4 SEQ ID NO: 388 caatgtggcaacagaaata 701720 SEQ ID NO: 1391 tattcttcttttccaattg 13826 13845 1 4 SEQ ID NO: 389tggcaacagaaatatccac 706 725 SEQ ID NO: 1392 gtggcttcccatattgcca 18871906 1 4 SEQ ID NO: 390 agagacctgggccagtgtg 729 748 SEQ ID NO: 1393cacattacatttggtctct 2930 2949 1 4 SEQ ID NO: 391 tgtgatcgcttcaagccca 744763 SEQ ID NO: 1394 tgggaaagccgccctcaca 5210 5229 1 4 SEQ ID NO: 392gtgatcgcttcaagcccat 745 764 SEQ ID NO: 1395 atgggaaagccgccctcac 52095228 1 4 SEQ ID NO: 393 cagcccacttgctctcatc 776 795 SEQ ID NO: 1396gatgctgaacagtgagctg 8144 8163 1 4 SEQ ID NO: 394 gctctcatcaaaggcatga 786805 SEQ ID NO: 1397 tcataacagtactgtgagc 10337 10356 1 4 SEQ ID NO: 395ccttgtcaactctgatcag 811 830 SEQ ID NO: 1398 ctgagtgggtttatcaagg 1244512464 1 4 SEQ ID NO: 396 cttgtcaactctgatcagc 812 831 SEQ ID NO: 1399gctgagtgggtttatcaag 12444 12463 1 4 SEQ ID NO: 397 agccatctgcaaggagcaa884 903 SEQ ID NO: 1400 ttgcaatgagctcatggct 3805 3824 1 4 SEQ ID NO: 398gccatctgcaaggagcaac 885 904 SEQ ID NO: 1401 gttgcaatgagctcatggc 38043823 1 4 SEQ ID NO: 399 cttcctgcctttctcctac 908 927 SEQ ID NO: 1402gtaggaataaatggagaag 9453 9472 1 4 SEQ ID NO: 400 ctttctcctacaagaataa 916935 SEQ ID NO: 1403 ttattgctgaatccaaaag 13648 13667 1 4 SEQ ID NO: 401gatcaacagccgcttcttt 989 1008 SEQ ID NO: 1404 aaagccatcactgatgatc 16611680 1 4 SEQ ID NO: 402 atcaacagccgcttctttg 990 1009 SEQ ID NO: 1405caaagccatcactgatgat 1660 1679 1 4 SEQ ID NO: 403 acagccgcttctttggtga 9941013 SEQ ID NO: 1406 tcacaaatcctttggctgt 9667 9686 1 4 SEQ ID NO: 404aagatgggcctcgcatttg 1023 1042 SEQ ID NO: 1407 caaaatagaagggaatctt 20692088 1 4 SEQ ID NO: 405 tgttttgaagactctccag 1082 1101 SEQ ID NO: 1408ctggtaactactttaaaca 5487 5506 1 4 SEQ ID NO: 406 ttgaagactctccaggaac1086 1105 SEQ ID NO: 1409 gttcaatgaatttattcaa 13184 13203 1 4 SEQ ID NO:407 aactgaaaaaactaaccat 1102 1121 SEQ ID NO: 1410 atggcattttttgcaagtt14006 14025 1 4 SEQ ID NO: 408 ctgaaaaaactaaccatct 1104 1123 SEQ ID NO:1411 agattgatgggcagttcag 4564 4583 1 4 SEQ ID NO: 409aaaactaaccatctctgag 1109 1128 SEQ ID NO: 1412 ctcaaagaatgactttttt 25702589 1 4 SEQ ID NO: 410 tgagcaaaatatccagaga 1124 1143 SEQ ID NO: 1413tctccagataaaaaactca 12201 12220 1 4 SEQ ID NO: 411 caataagctggttactgag1154 1173 SEQ ID NO: 1414 ctcagatcaaagttaattg 12265 12284 1 4 SEQ ID NO:412 tactgagctgagaggcctc 1166 1185 SEQ ID NO: 1415 gagggtagtcataacagta10329 10348 1 4 SEQ ID NO: 413 gcctcagtgatgaagcagt 1180 1199 SEQ ID NO:1416 actgttgactcaggaaggc 12572 12591 1 4 SEQ ID NO: 414agtcacatctctcttgcca 1196 1215 SEQ ID NO: 1417 tggccacatagcatggact 88588877 1 4 SEQ ID NO: 415 atctctcttgccacagctg 1202 1221 SEQ ID NO: 1418cagctgacctcatcgagat 2161 2180 1 4 SEQ ID NO: 416 tctctcttgccacagctga1203 1222 SEQ ID NO: 1419 tcagctgacctcatcgaga 2160 2179 1 4 SEQ ID NO:417 tgccacagctgattgaggt 1210 1229 SEQ ID NO: 1420 acctgcaccaaagctggca13955 13974 1 4 SEQ ID NO: 418 gccacagctgattgaggtg 1211 1230 SEQ ID NO:1421 caccaaaaaccccaatggc 11240 11259 1 4 SEQ ID NO: 419tcactttacaagccttggt 1240 1259 SEQ ID NO: 1422 accagatgctgaacagtga 81408159 1 4 SEQ ID NO: 420 cccttctgatagatgtggt 1324 1343 SEQ ID NO: 1423accacttacagctagaggg 10816 10835 1 4 SEQ ID NO: 421 gtcacctacctggtggccc1341 1360 SEQ ID NO: 1424 gggcgacctaagttgtgac 3431 3450 1 4 SEQ ID NO:422 ccttgtatgcgctgagcca 1432 1451 SEQ ID NO: 1425 tggctggtaacctaaaagg5578 5597 1 4 SEQ ID NO: 423 gacaaaccctacagggacc 1472 1491 SEQ ID NO:1426 ggtcctttatgattatgtc 12347 12366 1 4 SEQ ID NO: 424tgctaattacctgatggaa 1508 1527 SEQ ID NO: 1427 ttcccaaaagcagtcagca 99309949 1 4 SEQ ID NO: 425 tgactgcactggggatgaa 1538 1557 SEQ ID NO: 1428ttcaggtccatgcaagtca 10909 10928 1 4 SEQ ID NO: 426 actgcactggggatgaaga1540 1559 SEQ ID NO: 1429 tcttgaacacaaagtcagt 5999 6018 1 4 SEQ ID NO:427 atgaagattacacctattt 1552 1571 SEQ ID NO: 1430 aaatgaaagtaaagatcat8110 8129 1 4 SEQ ID NO: 428 accatggagcagttaactc 1602 1621 SEQ ID NO:1431 gagtaaaccaaaacttggt 9016 9035 1 4 SEQ ID NO: 429gcagttaactccagaactc 1610 1629 SEQ ID NO: 1432 gagttactgaaaaagctgc 1371913738 1 4 SEQ ID NO: 430 cagaactcaagtcttcaat 1621 1640 SEQ ID NO: 1433attggatatccaagatctg 1925 1944 1 4 SEQ ID NO: 431 caggctctgcggaaaatgg1695 1714 SEQ ID NO: 1434 ccatgacctccagctcctg 2477 2496 1 4 SEQ ID NO:432 ccaggaggttcttcttcag 1730 1749 SEQ ID NO: 1435 ctgaaatacaatgctctgg5511 5530 1 4 SEQ ID NO: 433 ggttcttcttcagactttc 1736 1755 SEQ ID NO:1436 gaaaaacttggaaacaacc 4431 4450 1 4 SEQ ID NO: 434tttccttgatgatgcttct 1751 1770 SEQ ID NO: 1437 agaatccagatacaagaaa 68856904 1 4 SEQ ID NO: 435 ggagataagcgactggctg 1773 1792 SEQ ID NO: 1438cagcatgcctagtttctcc 9944 9963 1 4 SEQ ID NO: 436 gctgcctatcttatgttga1788 1807 SEQ ID NO: 1439 tcaatatcaaaagcccagc 12037 12056 1 4 SEQ ID NO:437 actttgtggcttcccatat 1882 1901 SEQ ID NO: 1440 atatctggaaccttgaagt10729 10748 1 4 SEQ ID NO: 438 gccaatatcttgaactcag 1902 1921 SEQ ID NO:1441 ctgaactcagaaggatggc 13992 14011 1 4 SEQ ID NO: 439aatatcttgaactcagaag 1905 1924 SEQ ID NO: 1442 cttccattctgaatatatt 1337013389 1 4 SEQ ID NO: 440 ctcagaagaattggatatc 1916 1935 SEQ ID NO: 1443gataaaagattactttgag 7265 7284 1 4 SEQ ID NO: 441 aagaattggatatccaaga1921 1940 SEQ ID NO: 1444 tcttcaatttattcttctt 13817 13836 1 4 SEQ ID NO:442 agaattggatatccaagat 1922 1941 SEQ ID NO: 1445 atcttcaatttattcttct13816 13835 1 4 SEQ ID NO: 443 tggatatccaagatctgaa 1927 1946 SEQ ID NO:1446 ttcacataccagaattcca 8317 8336 1 4 SEQ ID NO: 444atatccaagatctgaaaaa 1930 1949 SEQ ID NO: 1447 tttttaaccagtcagatat 1017710196 1 4 SEQ ID NO: 445 tatccaagatctgaaaaag 1931 1950 SEQ ID NO: 1448ctttttaaccagtcagata 10176 10195 1 4 SEQ ID NO: 446 caagatctgaaaaagttag1935 1954 SEQ ID NO: 1449 ctaaattcccatggtcttg 4965 4984 1 4 SEQ ID NO:447 aagatctgaaaaagttagt 1936 1955 SEQ ID NO: 1450 actaaattcccatggtctt4964 4983 1 4 SEQ ID NO: 448 tgaaaaagttagtgaaaga 1942 1961 SEQ ID NO:1451 tctttctcgggaatattca 10622 10641 1 4 SEQ ID NO: 449tccaactgtcatggacttc 1982 2001 SEQ ID NO: 1452 gaagcacatatgaactgga 1393713956 1 4 SEQ ID NO: 450 tcagaaaattctctcggaa 1999 2018 SEQ ID NO: 1453ttcctttaacaattcctga 9493 9512 1 4 SEQ ID NO: 451 ttccatcacttgacccagc2044 2063 SEQ ID NO: 1454 gctgacatagggaatggaa 8433 8452 1 4 SEQ ID NO:452 cccagcctcagccaaaata 2057 2076 SEQ ID NO: 1455 tattctatccaagattggg7812 7831 1 4 SEQ ID NO: 453 agcctcagccaaaatagaa 2060 2079 SEQ ID NO:1456 ttctatccaagattgggct 7814 7833 1 4 SEQ ID NO: 454atcttatatttgatccaaa 2083 2102 SEQ ID NO: 1457 tttgaaaaacaaagcagat 1181311832 1 4 SEQ ID NO: 455 tcttatatttgatccaaat 2084 2103 SEQ ID NO: 1458attttttgcaagttaaaga 14011 14030 1 4 SEQ ID NO: 456 cttcctaaagaaagcatgc2109 2128 SEQ ID NO: 1459 gcatggcattatgatgaag 3606 3625 1 4 SEQ ID NO:457 ctaaagaaagcatgctgaa 2113 2132 SEQ ID NO: 1460 ttcagggtgtggagtttag5686 5705 1 4 SEQ ID NO: 458 taaagaaagcatgctgaaa 2114 2133 SEQ ID NO:1461 tttcttaaacattccttta 9482 9501 1 4 SEQ ID NO: 459gagattggcttggaaggaa 2175 2194 SEQ ID NO: 1462 ttccctccattaagttctc 1170111720 1 4 SEQ ID NO: 460 ctttgagccaacattggaa 2198 2217 SEQ ID NO: 1463ttccaatgaccaagaaaag 11060 11079 1 4 SEQ ID NO: 461 cagacagtgtcaacaaagc2245 2264 SEQ ID NO: 1464 gcttactggacgaactctg 6134 6153 1 4 SEQ ID NO:462 cagtgtcaacaaagctttg 2249 2268 SEQ ID NO: 1465 caaattcctggatacactg9849 9868 1 4 SEQ ID NO: 463 agtgtcaacaaagctttgt 2250 2269 SEQ ID NO:1466 acaagaatacgtctacact 4351 4370 1 4 SEQ ID NO: 464ctgatggtgtctctaaggt 2290 2309 SEQ ID NO: 1467 acctcggaacaatcctcag 33253344 1 4 SEQ ID NO: 465 tgatggtgtctctaaggtc 2291 2310 SEQ ID NO: 1468gacctgcgcaacgagatca 8823 8842 1 4 SEQ ID NO: 466 aaacatgagcaggatatgg2343 2362 SEQ ID NO: 1469 ccatgatctacatttgttt 6788 6807 1 4 SEQ ID NO:467 gaagctgattaaagatttg 2387 2406 SEQ ID NO: 1470 caaaaacattttcaacttc5279 5298 1 4 SEQ ID NO: 468 aaagatttgaaatccaaag 2397 2416 SEQ ID NO:1471 ctttaagttcagcatcttt 7606 7625 1 4 SEQ ID NO: 469gatgggtgcccgcactctg 2510 2529 SEQ ID NO: 1472 cagatttgaggattccatc 79757994 1 4 SEQ ID NO: 470 gggatcccccagatgattg 2532 2551 SEQ ID NO: 1473caatcacaagtcgattccc 9075 9094 1 4 SEQ ID NO: 471 ttttcttcactacatcttc2585 2604 SEQ ID NO: 1474 gaagtgtcagtggcaaaaa 10374 10393 1 4 SEQ ID NO:472 tcttcactacatcttcatg 2588 2607 SEQ ID NO: 1475 catggcattatgatgaaga3607 3626 1 4 SEQ ID NO: 473 tacatcttcatggagaatg 2595 2614 SEQ ID NO:1476 cattatggaggcccatgta 9437 9456 1 4 SEQ ID NO: 474ttcatggagaatgcctttg 2601 2620 SEQ ID NO: 1477 caaaatcaactttaatgaa 65996618 1 4 SEQ ID NO: 475 tcatggagaatgcctttga 2602 2621 SEQ ID NO: 1478tcaacacaatcttcaatga 13108 13127 1 4 SEQ ID NO: 476 tttgaactccccactggag2616 2635 SEQ ID NO: 1479 ctccccaggacctttcaaa 9834 9653 1 4 SEQ ID NO:477 ttgaactccccactggagc 2617 2636 SEQ ID NO: 1480 gctccccaggacctttcaa9833 9852 1 4 SEQ ID NO: 478 tgaactccccactggagct 2618 2637 SEQ ID NO:1481 agctccccaggacctttca 9832 9851 1 4 SEQ ID NO: 479cactggagctggattacag 2627 2646 SEQ ID NO: 1482 ctgtttctgagtcccagtg 93369355 1 4 SEQ ID NO: 480 actggagctggattacagt 2628 2647 SEQ ID NO: 1483actgtttctgagtcccagt 9335 9354 1 4 SEQ ID NO: 481 agttgcaaatatcttcatc2644 2663 SEQ ID NO: 1484 gatgatgccaaaatcaact 6591 6610 1 4 SEQ ID NO:482 gttgcaaatatcttcatct 2645 2664 SEQ ID NO: 1485 agatgatgccaaaatcaac6590 6609 1 4 SEQ ID NO: 483 aaatatcttcatctggagt 2650 2669 SEQ ID NO:1486 actcagaaggatggcattt 13996 14015 1 4 SEQ ID NO: 484taaaactggaagtagccaa 2695 2714 SEQ ID NO: 1487 ttggttacaggaggcttta 75927611 1 4 SEQ ID NO: 485 ggctgaactggtggcaaaa 2720 2739 SEQ ID NO: 1488ttttcttttcagcccagcc 9220 9239 1 4 SEQ ID NO: 486 tgtggagtttgtgacaaat2750 2769 SEQ ID NO: 1489 attttcaagcaaatgcaca 8530 8549 1 4 SEQ ID NO:487 ttgtgacaaatatgggcat 2758 2777 SEQ ID NO: 1490 atgcgtctaccttacacaa9513 9532 1 4 SEQ ID NO: 488 atgaacaccaacttcttcc 2811 2830 SEQ ID NO:1491 ggaagctgaagtttatcat 2869 2888 1 4 SEQ ID NO: 489cttccacgagtcgggtctg 2825 2844 SEQ ID NO: 1492 cagagctatcactgggaag 52275246 1 4 SEQ ID NO: 490 gagtcgggtctggaggctc 2832 2851 SEQ ID NO: 1493gagcttactggacgaactc 6132 6151 1 4 SEQ ID NO: 491 cctaaaagctgggaagctg2858 2877 SEQ ID NO: 1494 cagcctccccagccgtagg 12112 12131 1 4 SEQ ID NO:492 agctgggaagctgaagttt 2864 2883 SEQ ID NO: 1495 aaactgttaatttacagct5455 5474 1 4 SEQ ID NO: 493 ccagattagagctggaact 3106 3125 SEQ ID NO:1496 agtttccggggaaacctgg 12718 12737 1 4 SEQ ID NO: 494ggataccctgaagtttgta 3200 3219 SEQ ID NO: 1497 tacagtattctgaaaatcc 83858404 1 4 SEQ ID NO: 495 ctgaggctaccatgacatt 3244 3263 SEQ ID NO: 1498aatgagctcatggcttcag 3809 3828 1 4 SEQ ID NO: 496 tgtccagtgaagtccaaat3289 3308 SEQ ID NO: 1499 attttgagaggaatcgaca 6349 6368 1 4 SEQ ID NO:497 aattccggattttgatgtt 3305 3324 SEQ ID NO: 1500 aacacatgaatcacaaatt8930 8949 1 4 SEQ ID NO: 498 ttccggattttgatgttga 3307 3326 SEQ ID NO:1501 tcaaaacgagcttcaggaa 13199 13218 1 4 SEQ ID NO: 499cggaacaatcctcagagtt 3329 3348 SEQ ID NO: 1502 aacttgtacaactggtccg 42034222 1 4 SEQ ID NO: 500 tcctcagagttaatgatga 3337 3356 SEQ ID NO: 1503tcatcaattggttacagga 7585 7604 1 4 SEQ ID NO: 501 ctcaccctggacattcaga3384 3403 SEQ ID NO: 1504 tctgcagaacaatgctgag 12431 12450 1 4 SEQ ID NO:502 cattcagaacaagaaaatt 3395 3414 SEQ ID NO: 1505 aattgactttgtagaaatg8096 8115 1 4 SEQ ID NO: 503 actgaggtcgccctcatgg 3414 3433 SEQ ID NO:1506 ccatgcaagtcagcccagt 10916 10935 1 4 SEQ ID NO: 504ttatttccataccccgttt 3478 3497 SEQ ID NO: 1507 aaactgcctatattgataa 1387213891 1 4 SEQ ID NO: 505 gtttgcaagcagaagccag 3493 3512 SEQ ID NO: 1508ctggacttctcttcaaaac 5400 5419 1 4 SEQ ID NO: 506 tttgcaagcagaagccaga3494 3513 SEQ ID NO: 1509 tctgggtgtcgacagcaaa 5264 5283 1 4 SEQ ID NO:507 ttgcaagcagaagccagaa 3495 3514 SEQ ID NO: 1510 ttctgggtgtcgacagcaa5263 5282 1 4 SEQ ID NO: 508 ctgcttctccaaatggact 3546 3565 SEQ ID NO:1511 agtcaagattgatgggcag 4559 4578 1 4 SEQ ID NO: 509tgctacagcttatggctcc 3569 3588 SEQ ID NO: 1512 ggaggctttaagttcagca 76017620 1 4 SEQ ID NO: 510 acagcttatggctccacag 3573 3592 SEQ ID NO: 1513ctgtatagcaaattcctgt 5889 5908 1 4 SEQ ID NO: 511 tttccaagagggtggcatg3592 3611 SEQ ID NO: 1514 catggacttcttctggaaa 8869 8888 1 4 SEQ ID NO:512 ccaagagggtggcatggca 3595 3614 SEQ ID NO: 1515 tgcccagcaagcaagttgg9353 9372 1 4 SEQ ID NO: 513 gtggcatggcattatgatg 3603 3622 SEQ ID NO:1516 catccttaacaccttccac 8063 8082 1 4 SEQ ID NO: 514tgatgaagagaagattgaa 3617 3636 SEQ ID NO: 1517 ttcactgttcctgaaatca 78637882 1 4 SEQ ID NO: 515 gaagagaagattgaatttg 3621 3640 SEQ ID NO: 1518caaaaacattttcaacttc 5279 5298 1 4 SEQ ID NO: 516 gagaagattgaatttgaat3624 3643 SEQ ID NO: 1519 attcataatcccaactctc 8270 8289 1 4 SEQ ID NO:517 tttgaatggaacacaggca 3636 3655 SEQ ID NO: 1520 tgcctttgtgtacaccaaa11228 11247 1 4 SEQ ID NO: 518 aggcaccaatgtagatacc 3650 3669 SEQ ID NO:1521 ggtaacctaaaaggagcct 5583 5602 1 4 SEQ ID NO: 519caaaaaaatgacttccaat 3668 3687 SEQ ID NO: 1522 attgaagtacctacttttg 83588377 1 4 SEQ ID NO: 520 aaaaaaatgacttccaatt 3669 3688 SEQ ID NO: 1523aattgaagtacctactttt 8357 8376 1 4 SEQ ID NO: 521 aaaaaatgacttccaattt3670 3689 SEQ ID NO: 1524 aaatccaatctcctctttt 8398 8417 1 4 SEQ ID NO:522 cagagtccctcaaacagac 3752 3771 SEQ ID NO: 1525 gtctgtgggattccatctg4082 4101 1 4 SEQ ID NO: 523 aaattaatagttgcaatga 3795 3814 SEQ ID NO:1526 tcataagttcaatgaattt 13178 13197 1 4 SEQ ID NO: 524ttcaacctccagaacatgg 3891 3910 SEQ ID NO: 1527 ccattgaccagatgctgaa 81348153 1 4 SEQ ID NO: 525 tgggattgccagacttcca 3907 3926 SEQ ID NO: 1528tggaaatgggcctgcccca 8895 8914 1 4 SEQ ID NO: 526 cagtttgaaaattgagatt3986 4005 SEQ ID NO: 1529 aatcacaactcctccactg 9533 9552 1 4 SEQ ID NO:527 gaaaattgagattcctttg 3992 4011 SEQ ID NO: 1530 caaaactaccacacatttc13686 13705 1 4 SEQ ID NO: 528 tttgccttttggtggcaaa 4007 4026 SEQ ID NO:1531 tttgagaggaatcgacaaa 6351 6370 1 4 SEQ ID NO: 529ctccagagatctaaagatg 4028 4047 SEQ ID NO: 1532 catcaattggttacaggag 75867605 1 4 SEQ ID NO: 530 tctaaagatgttagagact 4037 4056 SEQ ID NO: 1533agtccttcatgtccctaga 10025 10044 1 4 SEQ ID NO: 531 ctgtgggattccatctgcc4084 4103 SEQ ID NO: 1534 ggcattttgaaaaaaacag 9727 9746 1 4 SEQ ID NO:532 atctgccatctcgagagtt 4096 4115 SEQ ID NO: 1535 aactctcaaaccctaagat8548 8567 1 4 SEQ ID NO: 533 tctcgagagttccaagtcc 4104 4123 SEQ ID NO:1536 ggacattcctctagcgaga 8207 8226 1 4 SEQ ID NO: 534agtccctacttttaccatt 4118 4137 SEQ ID NO: 1537 aatgaatacagccaggact 60786097 1 4 SEQ ID NO: 535 acttttaccattcccaagt 4125 4144 SEQ ID NO: 1538actttgtagaaatgaaagt 8101 8120 1 4 SEQ ID NO: 536 cattcccaagttgtatcaa4133 4152 SEQ ID NO: 1539 ttgaaggacttcaggaatg 12001 12020 1 4 SEQ ID NO:537 accacatgaaggctgactc 4276 4295 SEQ ID NO: 1540 gagtaaaccaaaacttggt9016 9035 1 4 SEQ ID NO: 538 tttcctacaatgtgcaagg 4309 4328 SEQ ID NO:1541 cctttaacaattcctgaaa 9495 9514 1 4 SEQ ID NO: 539ctggagaaacaacatatga 4330 4349 SEQ ID NO: 1542 tcattctgggtctttccag 1102711046 1 4 SEQ ID NO: 540 atcatgtgatgggtctcta 4370 4369 SEQ ID NO: 1543tagaattacagaaaatgat 6557 6576 1 4 SEQ ID NO: 541 catgtgatgggtctctacg4372 4391 SEQ ID NO: 1544 cgtaggcaccgtgggcatg 12125 12144 1 4 SEQ ID NO:542 ttctagattcgaatatcaa 4399 4418 SEQ ID NO: 1545 ttgatgatgctgtcaagaa7300 7319 1 4 SEQ ID NO: 543 tggggaccacagatgtctg 4491 4510 SEQ ID NO:1546 cagaattccagcttcccca 8326 8345 1 4 SEQ ID NO: 544ctaacactggccggctcaa 4636 4655 SEQ ID NO: 1547 ttgaggctattgatgttag 69766995 1 4 SEQ ID NO: 545 taacactggccggctcaat 4637 4656 SEQ ID NO: 1548attgaggctattgatgtta 6975 6994 1 4 SEQ ID NO: 546 aacactggccggctcaatg4638 4657 SEQ ID NO: 1549 cattgaggctattgatgtt 6974 6993 1 4 SEQ ID NO:547 ctggccggctcaatggaga 4642 4661 SEQ ID NO: 1550 tctccatctgcgctaccag12065 12084 1 4 SEQ ID NO: 548 agataacaggaagatatga 4705 4724 SEQ ID NO:1551 tcatctcctttcttcatct 10202 10221 1 4 SEQ ID NO: 549tccctcacctccacctctg 4737 4756 SEQ ID NO: 1552 cagatatatatctcaggga 81768195 1 4 SEQ ID NO: 550 agctgactttaaaatctga 4810 4829 SEQ ID NO: 1553tcaggctcttcagaaagct 7922 7941 1 4 SEQ ID NO: 551 ctgactttaaaatctgaca4812 4831 SEQ ID NO: 1554 tgtcaagataaacaatcag 8732 8751 1 4 SEQ ID NO:552 caagatggatatgaccttc 4865 4884 SEQ ID NO: 1555 gaagtagtactgcatcttg6835 6854 1 4 SEQ ID NO: 553 gctgcgttctgaatatcag 4901 4920 SEQ ID NO:1556 ctgagtcccagtgcccagc 9342 9361 1 4 SEQ ID NO: 554cgttctgaatatcaggctg 4905 4924 SEQ ID NO: 1557 cagcaagtacctgagaacg 86038622 1 4 SEQ ID NO: 555 aattcccatggtcttgagt 4968 4987 SEQ ID NO: 1558actcagatcaaagttaatt 12264 12283 1 4 SEQ ID NO: 556 tggtcttgagttaaatgct4976 4995 SEQ ID NO: 1559 agcacagtacgaaaaacca 10801 10820 1 4 SEQ ID NO:557 cttgagttaaatgctgaca 4980 4999 SEQ ID NO: 1560 tgtccctagaaatctcaag10034 10053 1 4 SEQ ID NO: 558 ttgagttaaatgctgacat 4981 5000 SEQ ID NO:1561 atgtccctagaaatctcaa 10033 10052 1 4 SEQ ID NO: 559tgagttaaatgctgacatc 4982 5001 SEQ ID NO: 1562 gatggaaccctctccctca 47254744 1 4 SEQ ID NO: 560 acttgaagtgtagtctcct 5086 5105 SEQ ID NO: 1563aggaaactcagatcaaagt 12259 12278 1 4 SEQ ID NO: 561 agtgtagtctcctggtgct5092 5111 SEQ ID NO: 1564 agcagccagtggcaccact 12506 12525 1 4 SEQ ID NO:562 gtgctggagaatgagctga 5106 5125 SEQ ID NO: 1565 tcagccaggtttatagcac7726 7745 1 4 SEQ ID NO: 563 ctggggcatctatgaaatt 5143 5162 SEQ ID NO:1566 aatttctgattaccaccag 13571 13590 1 4 SEQ ID NO: 564atggccgcttcagggaaca 5170 5189 SEQ ID NO: 1567 tgttttttggaaatgccat 86418660 1 4 SEQ ID NO: 565 ttcagtctggatgggaaag 5199 5218 SEQ ID NO: 1568ctttgacaggcattttgaa 9719 9738 1 4 SEQ ID NO: 566 ccatgattctgggtgtcga5257 5276 SEQ ID NO: 1569 tcgatgcacatacaaatgg 5830 5849 1 4 SEQ ID NO:567 aaaacattttcaacttcaa 5281 5300 SEQ ID NO: 1570 ttgatgttagagtgctttt6985 7004 1 4 SEQ ID NO: 568 cttaagctctcaaatgaca 5316 5335 SEQ ID NO:1571 tgtcctacaacaagttaag 7247 7266 1 4 SEQ ID NO: 569ttaagctctcaaatgacat 5317 5336 SEQ ID NO: 1572 atgtcctacaacaagttaa 72467265 1 4 SEQ ID NO: 570 catgatgggctcatatgct 5333 5352 SEQ ID NO: 1573agcatctttggctcacatg 7616 7635 1 4 SEQ ID NO: 571 tgggctcatatgctgaaat5338 5357 SEQ ID NO: 1574 atttatcaaaagaagccca 12934 12953 1 4 SEQ ID NO:572 actggacttctcttcaaaa 5399 5418 SEQ ID NO: 1575 ttttggcaagctatacagt8372 8391 1 4 SEQ ID NO: 573 acttctcttcaaaacttga 5404 5423 SEQ ID NO:1576 tcaattgggagagacaagt 6496 6515 1 4 SEQ ID NO: 574ctgacaagttttataagca 5437 5456 SEQ ID NO: 1577 tgctttgtgagtttatcag 96859704 1 4 SEQ ID NO: 575 aagttttataagcaaactg 5442 5461 SEQ ID NO: 1578cagtcatgtagaaaaactt 4421 4440 1 4 SEQ ID NO: 576 ctgttaatttacagctaca5458 5477 SEQ ID NO: 1579 tgtactggaaaacgtacag 6380 6399 1 4 SEQ ID NO:577 ttacagctacagccctatt 5466 5485 SEQ ID NO: 1580 aatattgatcaatttgtaa6417 6436 1 4 SEQ ID NO: 578 tctggtaactactttaaac 5486 5505 SEQ ID NO:1581 gtttgaaaaacaaagcaga 11812 11831 1 4 SEQ ID NO: 579tttaaacagtgacctgaaa 5498 5517 SEQ ID NO: 1582 tttcatttgaaagaataaa 70247043 1 4 SEQ ID NO: 580 ttaaacagtgacctgaaat 5499 5518 SEQ ID NO: 1583atttcaagcaagaacttaa 10426 10445 1 4 SEQ ID NO: 581 cagtgacctgaaatacaat5504 5523 SEQ ID NO: 1584 attggcgtggagcttactg 6123 6142 1 4 SEQ ID NO:582 tgtggctggtaacctaaaa 5576 5595 SEQ ID NO: 1585 ttttgctggagaagccaca10757 10776 1 4 SEQ ID NO: 583 ttatcagcaagctataaag 5649 5668 SEQ ID NO:1586 ctttgcactatgttcataa 12756 12775 1 4 SEQ ID NO: 584ggttcagggtgtggagttt 5684 5703 SEQ ID NO: 1587 aaacacctaagagtaaacc 90069025 1 4 SEQ ID NO: 585 attcagactcactgcattt 5767 5786 SEQ ID NO: 1588aaatgctgacatagggaat 8429 8448 1 4 SEQ ID NO: 586 ttcagactcactgcatttc5768 5787 SEQ ID NO: 1589 gaaatattatgaacttgaa 13304 13323 1 4 SEQ ID NO:587 tacaaatggcaatgggaaa 5840 5859 SEQ ID NO: 1590 tttcctaaagctggatgta11168 11187 1 4 SEQ ID NO: 588 gctgtatagcaaattcctg 5888 5907 SEQ ID NO:1591 caggtccatgcaagtcagc 10911 10930 1 4 SEQ ID NO: 589tgagcagacaggcacctgg 6035 6054 SEQ ID NO: 1592 ccagcttccccacatctca 83338352 1 4 SEQ ID NO: 590 ggcacctggaaactcaaga 6045 6064 SEQ ID NO: 1593tcttcgtgtttcaactgcc 11213 11232 1 4 SEQ ID NO: 591 tgaatacagccaggacttg6080 6099 SEQ ID NO: 1594 caagtaagtgctaggttca 9372 9391 1 4 SEQ ID NO:592 gaatacagccaggacttgg 6081 6100 SEQ ID NO: 1595 ccaacacttacttgaattc10660 10679 1 4 SEQ ID NO: 593 ctggacgaactctggctga 6139 6158 SEQ ID NO:1596 tcagaaagctaccttccag 7931 7950 1 4 SEQ ID NO: 594ttttactcagtgagcccat 6193 6212 SEQ ID NO: 1597 atggacttcttctggaaaa 88708889 1 4 SEQ ID NO: 595 gatgagagatgccgttgag 6233 6252 SEQ ID NO: 1598ctcatctcctttcttcatc 10201 10220 1 4 SEQ ID NO: 596 aattgttgcttttgtaaag6269 6288 SEQ ID NO: 1599 cttttctaaacttgaaatt 9056 9075 1 4 SEQ ID NO:597 cttttgtaaagtatgataa 6277 6296 SEQ ID NO: 1600 ttatgaacttgaagaaaag13310 13329 1 4 SEQ ID NO: 598 tttgtaaagtatgataaaa 6279 6298 SEQ ID NO:1601 ttttcacattagatgcaaa 8413 8432 1 4 SEQ ID NO: 599tccattaacctcccatttt 6312 6331 SEQ ID NO: 1602 aaaattgatgatatctgga 1071910738 1 4 SEQ ID NO: 600 ccattaacctcccattttt 6313 6332 SEQ ID NO: 1603aaaagggtcatggaaatgg 8885 8904 1 4 SEQ ID NO: 601 cttgcaagaatattttgag6338 6357 SEQ ID NO: 1604 ctcaattttgattttcaag 8520 8539 1 4 SEQ ID NO:602 agaatattttgagaggaat 6344 6363 SEQ ID NO: 1605 attccctccattaagttct11700 11719 1 4 SEQ ID NO: 603 attatagttgtactggaaa 6372 6391 SEQ ID NO:1606 tttcaagcaagaacttaat 10427 10446 1 4 SEQ ID NO: 604gaagcacatcaatattgat 6407 6426 SEQ ID NO: 1607 atcagttcagataaacttc 79918010 1 4 SEQ ID NO: 605 acatcaatattgatcaatt 6412 6431 SEQ ID NO: 1608aattccctgaagttgatgt 11479 11498 1 4 SEQ ID NO: 606 gaaaactcccacagcaagc6457 6476 SEQ ID NO: 1609 gctttctcttccacatttc 10052 10071 1 4 SEQ ID NO:607 ctgaattcattcaattggg 6486 6505 SEQ ID NO: 1610 cccatttacagatcttcag11363 11382 1 4 SEQ ID NO: 608 tgaattcattcaattggga 6487 6506 SEQ ID NO:1611 tcccatttacagatcttca 11362 11381 1 4 SEQ ID NO: 609aactgactgctctcacaaa 6532 6551 SEQ ID NO: 1612 tttgaggattccatcagtt 79797998 1 4 SEQ ID NO: 610 aaaagtatagaattacaga 6550 6569 SEQ ID NO: 1613tctggctccctcaactttt 9042 9061 1 4 SEQ ID NO: 611 atcaactttaatgaaaaac6603 6622 SEQ ID NO: 1614 gtttattgaaaatattgat 6803 6822 1 4 SEQ ID NO:612 tgatttgaaaatagctatt 6686 6705 SEQ ID NO: 1615 aatattattgatgaaatca6708 6727 1 4 SEQ ID NO: 613 atttgaaaatagctattgc 6688 6707 SEQ ID NO:1616 gcaagaacttaatggaaat 10433 10452 1 4 SEQ ID NO: 614attgctaatattattgatg 6702 6721 SEQ ID NO: 1617 catcacactgaataccaat 1015110170 1 4 SEQ ID NO: 615 gaaaaattaaaaagtcttg 6729 6748 SEQ ID NO: 1618caagagcttatgggatttc 11153 11172 1 4 SEQ ID NO: 616 actatcatatccgtgtaat6754 6773 SEQ ID NO: 1619 attactttgagaaattagt 7273 7292 1 4 SEQ ID NO:617 tattgattttaacaaaagt 6815 6834 SEQ ID NO: 1620 acttgacttcagagaaata11396 11415 1 4 SEQ ID NO: 618 ctgcagcagcttaagagac 6906 6925 SEQ ID NO:1621 gtcttcagtgaagctgcag 10691 10710 1 4 SEQ ID NO: 619aaaacaacacattgaggct 6965 6984 SEQ ID NO: 1622 agcctcacctcttactttt 1056310582 1 4 SEQ ID NO: 620 ttgagcatgtcaaacactt 7051 7070 SEQ ID NO: 1623aagtagctgagaaaatcaa 7096 7115 1 4 SEQ ID NO: 621 tttgaagtagctgagaaaa7092 7111 SEQ ID NO: 1624 ttttcacattagatgcaaa 8413 8432 1 4 SEQ ID NO:622 ttagtagagttggcccacc 7191 7210 SEQ ID NO: 1625 ggtggactcttgctgctaa7768 7787 1 4 SEQ ID NO: 623 tgaaggagactattcagaa 7219 7238 SEQ ID NO:1626 ttctcaattttgattttca 8518 8537 1 4 SEQ ID NO: 624gagactattcagaagctaa 7224 7243 SEQ ID NO: 1627 ttagccacagctctgtctc 1029310312 1 4 SEQ ID NO: 625 aattagttggatttattga 7285 7304 SEQ ID NO: 1628tcaagaagcttaatgaatt 7312 7331 1 4 SEQ ID NO: 626 gcttaatgaattatctttt7319 7338 SEQ ID NO: 1629 aaaacgagcttcaggaagc 13201 13220 1 4 SEQ ID NO:627 ttaacaaattccttgacat 7357 7376 SEQ ID NO: 1630 atgtcctacaacaagttaa7246 7265 1 4 SEQ ID NO: 628 aaattaaagtcatttgatt 7386 7405 SEQ ID NO:1631 aatcctttgacaggcattt 9715 9734 1 4 SEQ ID NO: 629gactcaatggtgaaattca 7456 7475 SEQ ID NO: 1632 tgaaattcaatcacaagtc 90689087 1 4 SEQ ID NO: 630 gaaattcaggctctggaac 7467 7486 SEQ ID NO: 1633gttctcaattttgattttc 8517 8536 1 4 SEQ ID NO: 631 actaccacaaaaagctgaa7484 7503 SEQ ID NO: 1634 ttcaggaactattgctagt 10637 10656 1 4 SEQ ID NO:632 ccaaaataaccttaatcat 7570 7589 SEQ ID NO: 1635 atgatttccctgaccttgg10942 10961 1 4 SEQ ID NO: 633 aaataaccttaatcatcaa 7573 7592 SEQ ID NO:1636 ttgaagtaaaagaaaattt 10741 10760 1 4 SEQ ID NO: 634tttaagttcagcatctttg 7607 7626 SEQ ID NO: 1637 caaatctggatttcttaaa 94729491 1 4 SEQ ID NO: 635 caggtttatagcacacttg 7731 7750 SEQ ID NO: 1638caagggttcactgttcctg 7857 7876 1 4 SEQ ID NO: 636 gttcactgttcctgaaatc7862 7881 SEQ ID NO: 1639 gattctcagatgagggaac 8914 8933 1 4 SEQ ID NO:637 cactgttcctgaaatcaag 7865 7884 SEQ ID NO: 1640 cttgaacacaaagtcagtg6000 6019 1 4 SEQ ID NO: 638 actgttcctgaaatcaaga 7866 7885 SEQ ID NO:1641 tcttgaacacaaagtcagt 5999 6018 1 4 SEQ ID NO: 639gcctgcctttgaagtcagt 7901 7920 SEQ ID NO: 1642 actgttgactcaggaaggc 1257212591 1 4 SEQ ID NO: 640 taacagatttgaggattcc 7972 7991 SEQ ID NO: 1643ggaagcttctcaagagtta 13214 13233 1 4 SEQ ID NO: 641 gttttccacaccagaattt8042 8061 SEQ ID NO: 1644 aaatttctctgctggaaac 9410 9429 1 4 SEQ ID NO:642 tcagaaccattgaccagat 8128 8147 SEQ ID NO: 1645 atctgcagaacaatgctga12430 12449 1 4 SEQ ID NO: 643 tagcgagaatcaccctgcc 8218 8237 SEQ ID NO:1646 ggcagcttctggcttgcta 12293 12312 1 4 SEQ ID NO: 644ccttaatgattttcaagtt 8291 8310 SEQ ID NO: 1647 aactgttgactcaggaagg 1257112590 1 4 SEQ ID NO: 645 acataccagaattccagct 8320 8339 SEQ ID NO: 1648agctgccagtccttcatgt 10018 10037 1 4 SEQ ID NO: 646 aatgctgacatagggaatg8430 8449 SEQ ID NO: 1649 cattaatcctgccatcatt 9997 10016 1 4 SEQ ID NO:647 atgctgacatagggaatgg 8431 8450 SEQ ID NO: 1650 ccatttgagatcacggcat9237 9256 1 4 SEQ ID NO: 648 aaccacctcagcaaacgaa 8450 8469 SEQ ID NO:1651 ttcgttttccattaaggtt 9283 9302 1 4 SEQ ID NO: 649agcaggtatcgcagcttcc 8468 8487 SEQ ID NO: 1652 ggaagtggccctgaatgct 1096410983 1 4 SEQ ID NO: 650 tgcacaactctcaaaccct 8543 8562 SEQ ID NO: 1653agggaaagagaagattgca 13493 13512 1 4 SEQ ID NO: 651 aggagtcagtgaagttctc8584 8603 SEQ ID NO: 1654 gagaacttactatcatcct 13780 13799 1 4 SEQ ID NO:652 tttttggaaatgccattga 8644 8663 SEQ ID NO: 1655 tcaatgaatttattcaaaa13186 13205 1 4 SEQ ID NO: 653 aatggagtgattgtcaaga 8721 8740 SEQ ID NO:1656 tcttttcagcccagccatt 9223 9242 1 4 SEQ ID NO: 654gtcaagataaacaatcagc 8733 8752 SEQ ID NO: 1657 gctgactttaaaatctgac 48114830 1 4 SEQ ID NO: 655 tccacaaattgaacatccc 8779 8798 SEQ ID NO: 1658gggatttcctaaagctgga 11164 11183 1 4 SEQ ID NO: 656 ttgaacatccccaaactgg8787 8806 SEQ ID NO: 1659 ccagtttccagggactcaa 12595 12614 1 4 SEQ ID NO:657 acatccccaaactggactt 8791 8810 SEQ ID NO: 1660 aagtcgattcccagcatgt9082 9101 1 4 SEQ ID NO: 658 acttctctagtcaggctga 8806 8825 SEQ ID NO:1661 tcagatggaaaaatgaagt 11002 11021 1 4 SEQ ID NO: 659tgaatcacaaattagtttc 8936 8955 SEQ ID NO: 1662 gaaagtccataatggttca 1280912828 1 4 SEQ ID NO: 660 agaaggacccctcacttcc 8960 8979 SEQ ID NO: 1663ggaagaagaggcagcttct 12284 12303 1 4 SEQ ID NO: 661 ttggactgtccaataagat8980 8999 SEQ ID NO: 1664 atctaaatgcagtagccaa 11626 11645 1 4 SEQ ID NO:662 actgtccaataagatcaat 8984 9003 SEQ ID NO: 1665 attgataaaaccatacagt13883 13902 1 4 SEQ ID NO: 663 ctgtccaataagatcaata 8985 9004 SEQ ID NO:1666 tattgataaaaccatacag 13882 13901 1 4 SEQ ID NO: 664gtttatgaatctggctccc 9033 9052 SEQ ID NO: 1667 gggaatctgatgaggaaac 1224712266 1 4 SEQ ID NO: 665 atgaatctggctccctcaa 9037 9056 SEQ ID NO: 1668ttgagttgcccaccatcat 11659 11678 1 4 SEQ ID NO: 666 ctcaacttttctaaacttg9051 9070 SEQ ID NO: 1669 caagatcgcagactttgag 11645 11664 1 4 SEQ ID NO:667 ctaaaggcatggcactgtt 9121 9140 SEQ ID NO: 1670 aacagaaacaatgcattag9741 9760 1 4 SEQ ID NO: 668 aaggcatggcactgtttgg 9124 9143 SEQ ID NO:1671 ccaagaaaaggcacacctt 11069 11088 1 4 SEQ ID NO: 669atccacaaacaatgaaggg 9254 9273 SEQ ID NO: 1672 ccctaacagatttgaggat 79697988 1 4 SEQ ID NO: 670 ggaatttgaaagttcgttt 9271 9290 SEQ ID NO: 1673aaacaaacacaggcattcc 9647 9666 1 4 SEQ ID NO: 671 aataactatgcactgtttc9324 9343 SEQ ID NO: 1674 gaaatactgttttcctatt 12828 12847 1 4 SEQ ID NO:672 gaaacaacgagaacattat 9424 9443 SEQ ID NO: 1675 ataaactgcaagatttttc13600 13619 1 4 SEQ ID NO: 673 ttcttgaaaacgacaaagc 9591 9610 SEQ ID NO:1676 gctttccaatgaccaagaa 11057 11076 1 4 SEQ ID NO: 674ataagaaaaacaaacacag 9640 9659 SEQ ID NO: 1677 ctgtgctttgtgagtttat 96829701 1 4 SEQ ID NO: 675 aaaacaaacacaggcattc 9646 9665 SEQ ID NO: 1678gaatttgaaagttcgtttt 9272 9291 1 4 SEQ ID NO: 676 gcattccatcacaaatcct9659 9678 SEQ ID NO: 1679 aggaagtggccctgaatgc 10963 10982 1 4 SEQ ID NO:677 tttgaaaaaaacagaaaca 9732 9751 SEQ ID NO: 1680 tgttgaaagatttatcaaa12925 12944 1 4 SEQ ID NO: 678 caatgcattagattttgtc 9749 9768 SEQ ID NO:1681 gacaagaaaaaggggattg 10271 10290 1 4 SEQ ID NO: 679caaagctgaaaaatctcag 9809 9828 SEQ ID NO: 1682 ctgagaacttcatcatttg 1143011449 1 4 SEQ ID NO: 680 cctggatacactgttccag 9855 9874 SEQ ID NO: 1683ctggacttctctagtcagg 8802 8821 1 4 SEQ ID NO: 681 gttgaagtgtctccattca9882 9901 SEQ ID NO: 1684 tgaatctggctccctcaac 9038 9057 1 4 SEQ ID NO:682 tttctccatcctaggttct 9956 9975 SEQ ID NO: 1685 agaatccagatacaagaaa6885 6904 1 4 SEQ ID NO: 683 ttctccatcctaggttctg 9957 9976 SEQ ID NO:1686 cagaatccagatacaagaa 6884 6903 1 4 SEQ ID NO: 684tcattagagctgccagtcc 10011 10030 SEQ ID NO: 1687 ggacagtgaaatattatga13297 13316 1 4 SEQ ID NO: 685 tgctgaactttttaaccag 10169 10188 SEQ IDNO: 1688 ctggatgtaaccaccagca 11178 11197 1 4 SEQ ID NO: 686ctcctttcttcatcttcat 10206 10225 SEQ ID NO: 1689 atgaagcttgctccaggag13764 13783 1 4 SEQ ID NO: 687 tgtcattgatgcactgcag 10226 10245 SEQ IDNO: 1690 ctgcgctaccagaaagaca 12072 12091 1 4 SEQ ID NO: 688tgatgcactgcagtacaaa 10232 10251 SEQ ID NO: 1691 tttgagttgcccaccatca11658 11677 1 4 SEQ ID NO: 689 agctctgtctctgagcaac 10301 10320 SEQ IDNO: 1692 gttgaccacaagcttagct 10539 10558 1 4 SEQ ID NO: 690agccgaaattccaattttg 10400 10419 SEQ ID NO: 1693 caaagctggcaccagggct13963 13982 1 4 SEQ ID NO: 691 ttgagaatgaatttcaagc 10416 10435 SEQ IDNO: 1694 gcttcaggaagcttctcaa 13208 13227 1 4 SEQ ID NO: 692aaacctactgtctcttcct 10461 10480 SEQ ID NO: 1695 aggaaggccaagccagttt12583 12602 1 4 SEQ ID NO: 693 tacttttccattgagtcat 10575 10594 SEQ IDNO: 1696 atgattatgtcaacaagta 12355 12374 1 4 SEQ ID NO: 694tcaggtccatgcaagtcag 10910 10929 SEQ ID NO: 1697 ctgacatcttaggcactga 49935012 1 4 SEQ ID NO: 695 atgcaagtcagcccagttc 10918 10937 SEQ ID NO: 1698gaactcagaaggatggcat 13994 14013 1 4 SEQ ID NO: 696 tgaatgctaacactaagaa10975 10994 SEQ ID NO: 1699 ttctcaattttgattttca 8518 8537 1 4 SEQ ID NO:697 agaagatcagatggaaaaa 10996 11015 SEQ ID NO: 1700 ttttctaaatggaacttct12165 12184 1 4 SEQ ID NO: 698 ggctattcattctccatcc 11256 11275 SEQ IDNO: 1701 ggatctaaatgcagtagcc 11624 11643 1 4 SEQ ID NO: 699aaagttttggctgataaat 11280 11299 SEQ ID NO: 1702 atttcttaaacattccttt 94819500 1 4 SEQ ID NO: 700 agttttggctgataaattc 11282 11301 SEQ ID NO: 1703gaatctggctccctcaact 9039 9058 1 4 SEQ ID NO: 701 ctgggctgaaactaaatga11308 11327 SEQ ID NO: 1704 tcattctgggtctttccag 11027 11046 1 4 SEQ IDNO: 702 cagagaaatacaaatctat 11405 11424 SEQ ID NO: 1705atagcatggacttcttctg 8865 8884 1 4 SEQ ID NO: 703 gaggtaaaattccctgaag11472 11491 SEQ ID NO: 1706 cttctggcttgctaacctc 12298 12317 1 4 SEQ IDNO: 704 cttttttgagataaccgtg 11537 11556 SEQ ID NO: 1707cacggagttactgaaaaag 13715 13734 1 4 SEQ ID NO: 705 gctggaattgtcattcctt11727 11746 SEQ ID NO: 1708 aaggcatctccacctcagc 12094 12113 1 4 SEQ IDNO: 706 gtgtataatgccacttgga 11787 11806 SEQ ID NO: 1709tccaagatgagatcaacac 13096 13115 1 4 SEQ ID NO: 707 attccacatgcagctcaac11851 11870 SEQ ID NO: 1710 gttgagaagccccaagaat 6246 6265 1 4 SEQ ID NO:708 tgaagaagatggcaaattt 11984 12003 SEQ ID NO: 1711 aaattctcttttcttttca9212 9231 1 4 SEQ ID NO: 709 atcaaaagcccagcgttca 12042 12061 SEQ ID NO:1712 tgaaagtcaagcatctgat 12661 12680 1 4 SEQ ID NO: 710gtgggcatggatatggatg 12135 12154 SEQ ID NO: 1713 catccttaacaccttccac 80638082 1 4 SEQ ID NO: 711 aaatggaacttctactaca 12171 12190 SEQ ID NO: 1714tgtaccataagccatattt 10080 10099 1 4 SEQ ID NO: 712 aaaaactcaccatattcaa12211 12230 SEQ ID NO: 1715 ttgatgttagagtgctttt 6985 7004 1 4 SEQ ID NO:713 ctgagaagaaatctgcaga 12420 12439 SEQ ID NO: 1716 tctgcacagaaatattcag13439 13458 1 4 SEQ ID NO: 714 acaatgctgagtgggttta 12439 12458 SEQ IDNO: 1717 taaatggagtctttattgt 14078 14097 1 4 SEQ ID NO: 715caatgctgagtgggtttat 12440 12459 SEQ ID NO: 1718 ataaatggagtctttattg14077 14096 1 4 SEQ ID NO: 716 ttaggcaaattgatgatat 12469 12488 SEQ IDNO: 1719 atattgtcagtgcctctaa 13384 13403 1 4 SEQ ID NO: 717ataaactaatagatgtaat 12889 12908 SEQ ID NO: 1720 attactatgaaaaatttat13633 13652 1 4 SEQ ID NO: 718 ccaactaatagaagataac 13031 13050 SEQ IDNO: 1721 gttattttgctaaacttgg 14044 14063 1 4 SEQ ID NO: 719ttaattatatccaagatga 13087 13106 SEQ ID NO: 1722 tcatcctctaattttttaa13792 13811 1 4 SEQ ID NO: 720 tttaaattgttgaaagaaa 13143 13162 SEQ IDNO: 1723 tttcatttgaaagaataaa 7024 7043 1 4 SEQ ID NO: 721aagttcaatgaatttattc 13182 13201 SEQ ID NO: 1724 gaataccaatgctgaactt10160 10179 1 4 SEQ ID NO: 722 ttgaagaaaagatagtcag 13318 13337 SEQ IDNO: 1725 ctgagagaagtgtcttcaa 12399 12418 1 4 SEQ ID NO: 723acttccattctgaatatat 13369 13388 SEQ ID NO: 1726 atatctggaaccttgaagt10729 10748 1 4 SEQ ID NO: 724 cacagaaatattcaggaat 13443 13462 SEQ IDNO: 1727 attccctgaagttgatgtg 11480 11499 1 4 SEQ ID NO: 725ccattgcgacgaagaaaat 13552 13571 SEQ ID NO: 1728 atttttattcctgccatgg10095 10114 1 4 SEQ ID NO: 726 tataaactgcaagattttt 13599 13618 SEQ IDNO: 1729 aaaattcaaactgcctata 13865 13884 1 4 SEQ ID NO: 727tctgattactatgaaaaat 13629 13648 SEQ ID NO: 1730 atttgtaagaaaatacaga 64286447 1 4 SEQ ID NO: 728 ggagttactgaaaaagctg 13718 13737 SEQ ID NO: 1731cagcatgcctagtttctcc 9944 9963 1 4 SEQ ID NO: 729 tgaagcttgctccaggaga13765 13784 SEQ ID NO: 1732 tctcctttcttcatcttca 10205 10224 1 4 SEQ IDNO: 730 tgaactggacctgcaccaa 13947 13966 SEQ ID NO: 1733ttggtagagcaagggttca 7848 7867 1 4 SEQ ID NO: 731 ttgctaaacttgggggagg14050 14069 SEQ ID NO: 1734 cctcctacagtggtggcaa 4222 4241 1 4 SEQ ID NO:732 gattcgaatatcaaattca 4404 4423 SEQ ID NO: 1735 tgaaaacgacaaagcaatc9595 9614 3 3 SEQ ID NO: 733 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO:1736 acttttctaaacttgaaat 9055 9074 3 3 SEQ ID NO: 734tctcggttgctgccgctga 25 44 SEQ ID NO: 1737 tcagcccagccatttgaga 9228 92472 3 SEQ ID NO: 735 gctgaggagcccgcccagc 39 58 SEQ ID NO: 1738gctggatgtaaccaccagc 11177 11196 2 3 SEQ ID NO: 736 ctggtctgtccaaaagatg219 238 SEQ ID NO: 1739 catcagaaccattgaccag 8126 8145 2 3 SEQ ID NO: 737ctgagagttccagtggagt 283 302 SEQ ID NO: 1740 actcaatggtgaaattcag 74577476 2 3 SEQ ID NO: 738 cagtgcaccctgaaagagg 396 415 SEQ ID NO: 1741cctcacttcctttggactg 8969 8988 2 3 SEQ ID NO: 739 ctctgaggagtttgctgca 464483 SEQ ID NO: 1742 tgcaaacttgacttcagag 11391 11410 2 3 SEQ ID NO: 740acatcaagaggggcatcat 574 593 SEQ ID NO: 1743 atgacgttcttgagcatgt 70427061 2 3 SEQ ID NO: 741 ctgatcagcagcagccagt 822 841 SEQ ID NO: 1744actggacttctctagtcag 8801 8820 2 3 SEQ ID NO: 742 ggacgctaagaggaagcat 857876 SEQ ID NO: 1745 atgcctacgttccatgtcc 11346 11365 2 3 SEQ ID NO: 743agctgttttgaagactctc 1079 1098 SEQ ID NO: 1746 gagaagtgtcttcaaagct 1240312422 2 3 SEQ ID NO: 744 tgaaaaaactaaccatctc 1105 1124 SEQ ID NO: 1747gagatcaacacaatcttca 13104 13123 2 3 SEQ ID NO: 745 ctgagctgagaggcctcag1168 1187 SEQ ID NO: 1748 ctgaattactgcacctcag 3027 3046 2 3 SEQ ID NO:746 tgaaacgtgtgcatgccaa 1303 1322 SEQ ID NO: 1749 ttggtagagcaagggttca7848 7867 2 3 SEQ ID NO: 747 ccttgtatgcgctgagcca 1432 1451 SEQ ID NO:1750 tggcactgtttggagaagg 9130 9149 2 3 SEQ ID NO: 748aggagctgctggacattgc 1492 1511 SEQ ID NO: 1751 gcaagtcagcccagttcct 1092010939 2 3 SEQ ID NO: 749 atttgattctgcgggtcat 1567 1586 SEQ ID NO: 1752atgaaaccaatgacaaaat 7420 7439 2 3 SEQ ID NO: 750 tccagaactcaagtcttca1619 1638 SEQ ID NO: 1753 tgaaatacaatgctctgga 5512 5531 2 3 SEQ ID NO:751 ggttcttcttcagactttc 1736 1755 SEQ ID NO: 1754 gaaataccaagtcaaaacc10447 10466 2 3 SEQ ID NO: 752 gttgatgaggagtccttca 1802 1821 SEQ ID NO:1755 tgaaaaagctgcaatcaac 13726 13745 2 3 SEQ ID NO: 753tccaagatctgaaaaagtt 1933 1952 SEQ ID NO: 1756 aactgcttctccaaatgga 35443563 2 3 SEQ ID NO: 754 agttagtgaaagaagttct 1948 1967 SEQ ID NO: 1757agaattcataatcccaact 8267 8286 2 3 SEQ ID NO: 755 gaagggaatcttatatttg2076 2095 SEQ ID NO: 1758 caaaacctactgtctcttc 10459 10478 2 3 SEQ ID NO:756 ggaagctctttttgggaag 2213 2232 SEQ ID NO: 1759 cttcacataccagaattcc8316 8335 2 3 SEQ ID NO: 757 tggaataatgctcagtgtt 2366 2385 SEQ ID NO:1760 aacaaacacaggcattcca 9648 9667 2 3 SEQ ID NO: 758gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1761 cttcatgtccctagaaatc 1002910048 2 3 SEQ ID NO: 759 tccaaagaagtcccggaag 2409 2428 SEQ ID NO: 1762cttcagcctgctttctgga 4943 4962 2 3 SEQ ID NO: 760 aggaagggctcaaagaatg2562 2581 SEQ ID NO: 1763 cattagagctgccagtcct 10012 10031 2 3 SEQ ID NO:761 agaatgacttttttcttca 2575 2594 SEQ ID NO: 1764 tgaagatgacgacttttct12152 12171 2 3 SEQ ID NO: 762 tttgtgacaaatatgggca 2757 2776 SEQ ID NO:1765 tgccagtttgaaaaacaaa 11807 11826 2 3 SEQ ID NO: 763ctgaggctaccatgacatt 3244 3263 SEQ ID NO: 1766 aatgtcagctcttgttcag 1089510914 2 3 SEQ ID NO: 764 gtagataccaaaaaaatga 3660 3679 SEQ ID NO: 1767tcatttgccctcaacctac 11442 11461 2 3 SEQ ID NO: 765 aaatgacttccaatttccc3673 3692 SEQ ID NO: 1768 gggaactgttgaaagattt 12919 12938 2 3 SEQ ID NO:766 atgacttccaatttccctg 3675 3694 SEQ ID NO: 1769 caggagaacttactatcat13777 13796 2 3 SEQ ID NO: 767 atctgccatctcgagagtt 4096 4115 SEQ ID NO:1770 aactcctccactgaaagat 9539 9558 2 3 SEQ ID NO: 768atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 1771 acttccgtttaccagaaat 82398258 2 3 SEQ ID NO: 769 gcagagcttggcctctctg 5127 5146 SEQ ID NO: 1772cagagctttctgccactgc 13510 13529 2 3 SEQ ID NO: 770 atatgctgaaatgaaattt5345 5364 SEQ ID NO: 1773 aaattcaaactgcctatat 13866 13885 2 3 SEQ ID NO:771 tcaaaacttgacaacattt 5412 5431 SEQ ID NO: 1774 aaatacttccacaaattga8772 8791 2 3 SEQ ID NO: 772 cagtgacctgaaatacaat 5504 5523 SEQ ID NO:1775 attgaacatccccaaactg 8786 8805 2 3 SEQ ID NO: 773tacaaatggcaatgggaaa 5840 5859 SEQ ID NO: 1776 tttcaactgcctttgtgta 1122111240 2 3 SEQ ID NO: 774 cttttgtaaagtatgataa 6277 6296 SEQ ID NO: 1777ttattgctgaatccaaaag 13648 13667 2 3 SEQ ID NO: 775 ttgtaaagtatgataaaaa6280 6299 SEQ ID NO: 1778 ttttcaagcaaatgcacaa 8531 8550 2 3 SEQ ID NO:776 tccattaacctcccatttt 6312 6331 SEQ ID NO: 1779 aaaagaaaattttgctgga10748 10767 2 3 SEQ ID NO: 777 gattatctgaattcattca 6480 6499 SEQ ID NO:1780 tgaagtagaccaacaaatc 7154 7173 2 3 SEQ ID NO: 778aattgggagagacaagttt 6498 6517 SEQ ID NO: 1781 aaactaaatgatctaaatt 1131611335 2 3 SEQ ID NO: 779 atttgaaaatagctattgc 6688 6707 SEQ ID NO: 1782gcaatttctgcacagaaat 13433 13452 2 3 SEQ ID NO: 780 tgagcatgtcaaacacttt7052 7071 SEQ ID NO: 1783 aaagccattcagtctctca 12963 12982 2 3 SEQ ID NO:781 ttgaagatgttaacaaatt 7348 7367 SEQ ID NO: 1784 aattccatatgaaagtcaa12652 12671 2 3 SEQ ID NO: 782 acttgtcacctacatttct 7745 7764 SEQ ID NO:1785 agaatattttgatccaagt 13268 13287 2 3 SEQ ID NO: 783gttttccacaccagaattt 8042 8061 SEQ ID NO: 1786 aaatctggatttcttaaac 94739492 2 3 SEQ ID NO: 784 ataagtacaaccaaaattt 9397 9416 SEQ ID NO: 1787aaataaatggagtctttat 14075 14094 2 3 SEQ ID NO: 785 cgggacctgcggggctgag 019 SEQ ID NO: 1788 ctcagttaactgtgtcccg 11563 11582 1 3 SEQ ID NO: 786agtgcccttctcggttgct 17 36 SEQ ID NO: 1789 agcatctgattgactcact 1267012689 1 3 SEQ ID NO: 787 gctgaggagcccgcccagc 39 58 SEQ ID NO: 1790gctgattgaggtgtccagc 1217 1236 1 3 SEQ ID NO: 788 gaggagcccgcccagccag 4261 SEQ ID NO: 1791 ctggatcacagagtccctc 3744 3763 1 3 SEQ ID NO: 789gggccgcgaggccgaggcc 64 83 SEQ ID NO: 1792 ggccctgatccccgagccc 1355 13741 3 SEQ ID NO: 790 ccaggccgcagcccaggag 81 100 SEQ ID NO: 1793ctcccggagccaaggctgg 2674 2693 1 3 SEQ ID NO: 791 ggagccgccccaccgcagc 96115 SEQ ID NO: 1794 gctgttttgaagactctcc 1080 1099 1 3 SEQ ID NO: 792gaagaggaaatgctggaaa 192 211 SEQ ID NO: 1795 tttcaagttcctgaccttc 83018320 1 3 SEQ ID NO: 793 caaaagatgcgacccgatt 229 248 SEQ ID NO: 1796aatcttattggggattttg 7077 7096 1 3 SEQ ID NO: 794 attcaagcacctccggaag 245264 SEQ ID NO: 1797 cttccacatttcaaggaat 10059 10078 1 3 SEQ ID NO: 795gttccagtggagtccctgg 289 308 SEQ ID NO: 1798 ccagcaagtacctgagaac 86028621 1 3 SEQ ID NO: 796 gactgctgattcaagaagt 308 327 SEQ ID NO: 1799acttgaagaaaagatagtc 13316 13335 1 3 SEQ ID NO: 797 gtgccaccaggatcaactg325 344 SEQ ID NO: 1800 cagtgaagctgcagggcac 10696 10715 1 3 SEQ ID NO:798 gatcaactgcaaggttgag 335 354 SEQ ID NO: 1801 ctcacctccacctctgatc 47404759 1 3 SEQ ID NO: 799 actgcaaggttgagctgga 340 359 SEQ ID NO: 1802tccactcacatcctccagt 1281 1300 1 3 SEQ ID NO: 800 ccagctctgcagcttcatc 365384 SEQ ID NO: 1803 gatgtggtcacctacctgg 1335 1354 1 3 SEQ ID NO: 801agcttcatcctgaagacca 375 394 SEQ ID NO: 1804 tggtgctggagaatgagct 51045123 1 3 SEQ ID NO: 802 cttcatcctgaagaccagc 377 396 SEQ ID NO: 1805gctggagtaaaactggaag 2688 2707 1 3 SEQ ID NO: 803 ccagccagtgcaccctgaa 391410 SEQ ID NO: 1806 ttcaagatgactgcactgg 1531 1550 1 3 SEQ ID NO: 804cagtgcaccctgaaagagg 396 415 SEQ ID NO: 1807 cctcacagagctatcactg 52225241 1 3 SEQ ID NO: 805 tggcttcaaccctgagggc 419 438 SEQ ID NO: 1808gcccactggtcgcctgcca 3525 3544 1 3 SEQ ID NO: 806 cttcaaccctgagggcaaa 422441 SEQ ID NO: 1809 tttgagccaacattggaag 2199 2218 1 3 SEQ ID NO: 807ttcaaccctgagggcaaag 423 442 SEQ ID NO: 1810 ctttgacaggcattttgaa 97199738 1 3 SEQ ID NO: 808 cttgctgaagaaaaccaag 443 462 SEQ ID NO: 1811cttgaaattcaatcacaag 9066 9085 1 3 SEQ ID NO: 809 tgctgaagaaaaccaagaa 445464 SEQ ID NO: 1812 ttctgctgccttatcagca 5639 5658 1 3 SEQ ID NO: 810ttgctgcagccatgtccag 475 494 SEQ ID NO: 1813 ctggtcagtttgcaagcaa 29963015 1 3 SEQ ID NO: 811 tgctgcagccatgtccagg 476 495 SEQ ID NO: 1814cctggtcagtttgcaagca 2995 3014 1 3 SEQ ID NO: 812 agccatgtccaggtatgag 482501 SEQ ID NO: 1815 ctcacatcctccagtggct 1285 1304 1 3 SEQ ID NO: 813agctcaagctggccattcc 499 518 SEQ ID NO: 1816 ggaactaccacaaaaagct 74817500 1 3 SEQ ID NO: 814 agaagggaagcaggttttc 518 537 SEQ ID NO: 1817gaaatcttcaatttattct 13813 13832 1 3 SEQ ID NO: 815 aagggaagcaggttttcct520 539 SEQ ID NO: 1818 aggacaccaaaataacctt 7564 7583 1 3 SEQ ID NO: 816agaaagatgaacctactta 547 566 SEQ ID NO: 1619 taagaactttgccacttct 48444863 1 3 SEQ ID NO: 817 atcctgaacatcaagaggg 567 586 SEQ ID NO: 1820ccctaacagatttgaggat 7969 7988 1 3 SEQ ID NO: 818 tcctgaacatcaagagggg 568587 SEQ ID NO: 1821 cccctaacagatttgagga 7968 7987 1 3 SEQ ID NO: 819ctgaacatcaagaggggca 570 589 SEQ ID NO: 1822 tgcctgcctttgaagtcag 79007919 1 3 SEQ ID NO: 820 aacatcaagaggggcatca 573 592 SEQ ID NO: 1823tgataaaaaccaagatgtt 6290 6309 1 3 SEQ ID NO: 821 acatcaagaggggcatcat 574593 SEQ ID NO: 1824 atgataaaaaccaagatgt 6289 6308 1 3 SEQ ID NO: 822tcatttctgccctcctggt 589 608 SEQ ID NO: 1825 accaccagtttgtagatga 74057424 1 3 SEQ ID NO: 823 ttcccccagagacagaaga 607 626 SEQ ID NO: 1826tcttccacatttcaaggaa 10058 10077 1 3 SEQ ID NO: 824 gaagaagccaagcaagtgt621 640 SEQ ID NO: 1827 acaccttccacattccttc 8071 8090 1 3 SEQ ID NO: 825ttgtttctggataccgtgt 639 658 SEQ ID NO: 1828 acactaaatacttccacaa 87678786 1 3 SEQ ID NO: 826 tgtatggaaactgctccac 655 674 SEQ ID NO: 1829gtggaggcaacacattaca 2920 2939 1 3 SEQ ID NO: 827 aaactgctccactcacttt 662681 SEQ ID NO: 1830 aaagaaacagcatttgttt 4532 4551 1 3 SEQ ID NO: 828actcactttaccgtcaaga 672 691 SEQ ID NO: 1831 tcttacttttccattgagt 1057210591 1 3 SEQ ID NO: 829 ctttaccgtcaagacgagg 677 696 SEQ ID NO: 1832cctccagctcctgggaaag 2483 2502 1 3 SEQ ID NO: 830 ttaccgtcaagacgaggaa 679698 SEQ ID NO: 1833 ttcctaaagctggatgtaa 11169 11185 1 3 SEQ ID NO: 831acgaggaagggcaatgtgg 690 709 SEQ ID NO: 1834 ccacaagtcatcatctcgt 59565975 1 3 SEQ ID NO: 832 cgaggaagggcaatgtggc 691 710 SEQ ID NO: 1835gccagaagtgagatcctcg 3507 3526 1 3 SEQ ID NO: 833 gaggaagggcaatgtggca 692711 SEQ ID NO: 1836 tgccagtctccatgacctc 2468 2487 1 3 SEQ ID NO: 834ggaagggcaatgtggcaac 694 713 SEQ ID NO: 1837 gttgctcttaaggacttcc 1335613375 1 3 SEQ ID NO: 835 gaagggcaatgtggcaaca 695 714 SEQ ID NO: 1838tgttgatgaggagtccttc 1801 1820 1 3 SEQ ID NO: 836 caggcatcagcccacttgc 769788 SEQ ID NO: 1839 gcaagtctttcctggcctg 3011 3030 1 3 SEQ ID NO: 837aggcatcagcccacttgct 770 789 SEQ ID NO: 1840 agcaagtctttcctggcct 30103029 1 3 SEQ ID NO: 838 tcagcccacttgctctcat 775 794 SEQ ID NO: 1841atgaaagtcaagcatctga 12660 12679 1 3 SEQ ID NO: 839 gtcaactctgatcagcagc815 834 SEQ ID NO: 1842 gctgactttaaaatctgac 4811 4830 1 3 SEQ ID NO: 840ggacgctaagaggaagcat 857 876 SEQ ID NO: 1843 atgcactgtttctgagtcc 93319350 1 3 SEQ ID NO: 841 aaggagcaacacctcttcc 894 913 SEQ ID NO: 1844ggaatatcttagcatcctt 13457 13476 1 3 SEQ ID NO: 842 aggagcaacacctcttcct895 914 SEQ ID NO: 1845 aggaatatcttagcatcct 13456 13475 1 3 SEQ ID NO:843 caacacctcttcctgcctt 900 919 SEQ ID NO: 1846 aaggctgactctgtggttg 42844303 1 3 SEQ ID NO: 844 aacacctcttcctgccttt 901 920 SEQ ID NO: 1847aaagcaggccgaagctgtt 1067 1086 1 3 SEQ ID NO: 845 acaagaataagtatgggat 925944 SEQ ID NO: 1848 atccatgatctacatttgt 6786 6805 1 3 SEQ ID NO: 846caagaataagtatgggatg 926 945 SEQ ID NO: 1849 catcactttacaagccttg 12381257 1 3 SEQ ID NO: 847 tagcacaagtgacacagac 946 965 SEQ ID NO: 1850gtctcttcgttctatgcta 4584 4603 1 3 SEQ ID NO: 848 agcacaagtgacacagact 947966 SEQ ID NO: 1851 agtctcttcgttctatgct 4583 4602 1 3 SEQ ID NO: 849gcacaagtgacacagactt 948 967 SEQ ID NO: 1852 aagtgtagtctcctggtgc 50915110 1 3 SEQ ID NO: 850 aacttgaagacacaccaaa 970 989 SEQ ID NO: 1853tttgaggattccatcagtt 7979 7998 1 3 SEQ ID NO: 851 gcttctttggtgaaggtac1000 1019 SEQ ID NO: 1854 gtacctacttttggcaagc 8364 8383 1 3 SEQ ID NO:852 ctttggtgaaggtactaag 1004 1023 SEQ ID NO: 1855 cttatgggatttcctaaag11159 11178 1 3 SEQ ID NO: 853 tactaagaagatgggcctc 1016 1035 SEQ ID NO:1856 gagggtagtcataacagta 10329 10348 1 3 SEQ ID NO: 854tttgagagcaccaaatcca 1038 1057 SEQ ID NO: 1857 tggaagtgtcagtggcaaa 1037210391 1 3 SEQ ID NO: 855 agagcaccaaatccacatc 1042 1061 SEQ ID NO: 1858gatggatatgaccttctct 4868 4887 1 3 SEQ ID NO: 856 agctgttttgaagactctc1079 1098 SEQ ID NO: 1859 gagaacatactgggcagct 5872 5891 1 3 SEQ ID NO:857 tgaaaaaactaaccatctc 1105 1124 SEQ ID NO: 1860 gagaaaatcaatgccttca7104 7123 1 3 SEQ ID NO: 858 gaaaaaactaaccatctct 1106 1125 SEQ ID NO:1861 agagccaggtcgagctttc 11044 11063 1 3 SEQ ID NO: 859tctgagcaaaatatccaga 1122 1141 SEQ ID NO: 1862 tctgatgaggaaactcaga 1225212271 1 3 SEQ ID NO: 860 tctcttcaataagctggtt 1148 1167 SEQ ID NO: 1863aacctcccattttttgaga 6318 6337 1 3 SEQ ID NO: 861 ctgagctgagaggcctcag1168 1187 SEQ ID NO: 1864 ctgatccccgagccctcag 1359 1378 1 3 SEQ ID NO:862 tgaagcagtcacatctctc 1190 1209 SEQ ID NO: 1865 gagaaaatcaatgccttca7104 7123 1 3 SEQ ID NO: 863 aagcagtcacatctctctt 1192 1211 SEQ ID NO:1866 aagaggcagcttctggctt 12289 12308 1 3 SEQ ID NO: 864ctctcttgccacagctgat 1204 1223 SEQ ID NO: 1867 atcaaaagaagcccaagag 1293812957 1 3 SEQ ID NO: 865 tcttgccacagctgattga 1207 1226 SEQ ID NO: 1868tcaaagttaattgggaaga 12271 12290 1 3 SEQ ID NO: 866 cttgccacagctgattgag1208 1227 SEQ ID NO: 1869 ctcaattttgattttcaag 8520 8539 1 3 SEQ ID NO:867 tgaggtgtccagccccatc 1223 1242 SEQ ID NO: 1870 gatggaaccctctccctca4725 4744 1 3 SEQ ID NO: 868 tcagtgtggacagcctcag 1259 1278 SEQ ID NO:1871 ctgacatcttaggcactga 4993 5012 1 3 SEQ ID NO: 869acatcctccagtggctgaa 1288 1307 SEQ ID NO: 1872 ttcagaagctaagcaatgt 72317250 1 3 SEQ ID NO: 870 gcacagcagctgcgagaga 1377 1396 SEQ ID NO: 1873tctctgaaagacaacgtgc 12315 12334 1 3 SEQ ID NO: 871 cagcagctgcgagagatct1380 1399 SEQ ID NO: 1874 agataacattaaacagctg 13043 13062 1 3 SEQ ID NO:872 gcgagggatcagcgcagcc 1407 1426 SEQ ID NO: 1875 ggctcaacacagacatcgc5710 5729 1 3 SEQ ID NO: 873 aagacaaaccctacaggga 1470 1489 SEQ ID NO:1876 tcccagaaaacctcttctt 3928 3947 1 3 SEQ ID NO: 874caggagctgctggacattg 1491 1510 SEQ ID NO: 1877 caatggagagtccaacctg 46524671 1 3 SEQ ID NO: 875 aggagctgctggacattgc 1492 1511 SEQ ID NO: 1878gcaagggttcactgttcct 7856 7875 1 3 SEQ ID NO: 876 ctgctggacattgctaatt1497 1516 SEQ ID NO: 1879 aattgggaagaagaggcag 12279 12298 1 3 SEQ ID NO:877 gattacacctatttgattc 1557 1576 SEQ ID NO: 1880 gaatattttgagaggaatc6345 6364 1 3 SEQ ID NO: 878 atttgattctgcgggtcat 1567 1586 SEQ ID NO:1881 atgaagtagaccaacaaat 7153 7172 1 3 SEQ ID NO: 879tctgcgggtcattggaaat 1574 1593 SEQ ID NO: 1882 atttgtaagaaaatacaga 64286447 1 3 SEQ ID NO: 880 aaccatggagcagttaact 1601 1620 SEQ ID NO: 1883agtttctccatcctaggtt 9954 9973 1 3 SEQ ID NO: 881 ggagcagttaactccagaa1607 1626 SEQ ID NO: 1884 ttctgaaaatccaatctcc 8392 8411 1 3 SEQ ID NO:882 actccagaactcaagtctt 1617 1636 SEQ ID NO: 1885 aagatcgcagactttgagt11646 11665 1 3 SEQ ID NO: 883 tccagaactcaagtcttca 1619 1638 SEQ ID NO:1886 tgaactcagaagaattgga 1912 1931 1 3 SEQ ID NO: 884aagtacaaagccatcactg 1655 1674 SEQ ID NO: 1887 cagtcatgtagaaaaactt 44214440 1 3 SEQ ID NO: 885 gccatcactgatgatccag 1664 1683 SEQ ID NO: 1888ctggaactctctccatggc 10875 10894 1 3 SEQ ID NO: 886 ccatcactgatgatccaga1665 1684 SEQ ID NO: 1889 tctgaactcagaaggatgg 13991 14010 1 3 SEQ ID NO:887 atccagaaagctgccatcc 1677 1696 SEQ ID NO: 1890 ggatttcctaaagctggat11165 11184 1 3 SEQ ID NO: 888 cagaaagctgccatccagg 1680 1699 SEQ ID NO:1891 cctgaaatacaatgctctg 5510 5529 1 3 SEQ ID NO: 889acaaggaccaggaggttct 1723 1742 SEQ ID NO: 1892 agaaacagcatttgtttgt 45344553 1 3 SEQ ID NO: 890 aggaccaggaggttcttct 1726 1745 SEQ ID NO: 1893agaagctaagcaatgtcct 7234 7253 1 3 SEQ ID NO: 891 accaggaggttcttcttca1729 1748 SEQ ID NO: 1894 tgaaggctgactctgtggt 4282 4301 1 3 SEQ ID NO:892 tcttcagactttccttgat 1742 1761 SEQ ID NO: 1895 atcaggaagggctcaaaga2559 2578 1 3 SEQ ID NO: 893 ttcagactttccttgatga 1744 1763 SEQ ID NO:1896 tcattactcctgggctgaa 11299 11318 1 3 SEQ ID NO: 894gttgatgaggagtccttca 1802 1821 SEQ ID NO: 1897 tgaatctggctccctcaac 90389057 1 3 SEQ ID NO: 895 cttcacaggcagatattaa 1816 1835 SEQ ID NO: 1898ttaatcgagaggtatgaag 7140 7159 1 3 SEQ ID NO: 896 ttcacaggcagatattaac1817 1836 SEQ ID NO: 1899 gttaatcgagaggtatgaa 7139 7158 1 3 SEQ ID NO:897 ggcagatattaacaaaatt 1823 1842 SEQ ID NO: 1900 aattgcattagatgatgcc6581 6600 1 3 SEQ ID NO: 898 atattaacaaaattgtcca 1828 1847 SEQ ID NO:1901 tggagtttgtgacaaatat 2752 2771 1 3 SEQ ID NO: 899acaaaattgtccaaattct 1834 1853 SEQ ID NO: 1902 agaaacagcatttgtttgt 45344553 1 3 SEQ ID NO: 900 gagcaagtgaagaactttg 1869 1888 SEQ ID NO: 1903caaatgacatgatgggctc 5326 5345 1 3 SEQ ID NO: 901 gtgaagaactttgtggctt1875 1894 SEQ ID NO: 1904 aagcatctgattgactcac 12669 12688 1 3 SEQ ID NO:902 agaactttgtggcttccca 1879 1898 SEQ ID NO: 1905 tgggcctgccccagattct8901 8920 1 3 SEQ ID NO: 903 tttgtggcttcccatattg 1884 1903 SEQ ID NO:1906 caataagatcaatagcaaa 8990 9009 1 3 SEQ ID NO: 904tggcttcccatattgccaa 1888 1907 SEQ ID NO: 1907 ttggctcacatgaaggcca 76237642 1 3 SEQ ID NO: 905 ttcccatattgccaatatc 1892 1911 SEQ ID NO: 1908gatatacactagggaggaa 12737 12756 1 3 SEQ ID NO: 906 tcccatattgccaatatct1893 1912 SEQ ID NO: 1909 agatcaaagttaattggga 12268 12287 1 3 SEQ ID NO:907 ttgccaatatcttgaactc 1900 1919 SEQ ID NO: 1910 gagtcccagtgcccagcaa9344 9363 1 3 SEQ ID NO: 908 ttggatatccaagatctga 1926 1945 SEQ ID NO:1911 tcagtataagtacaaccaa 9392 9411 1 3 SEQ ID NO: 909tccaagatctgaaaaagtt 1933 1952 SEQ ID NO: 1912 aacttccaactgtcatgga 19781997 1 3 SEQ ID NO: 910 ctgaaaaagttagtgaaag 1941 1960 SEQ ID NO: 1913ctttgaagtcagtcttcag 7907 7926 1 3 SEQ ID NO: 911 agttagtgaaagaagttct1948 1967 SEQ ID NO: 1914 agaatctcaacttccaact 1970 1989 1 3 SEQ ID NO:912 aatctcaacttccaactgt 1972 1991 SEQ ID NO: 1915 acaggggtcctttatgatt12342 12361 1 3 SEQ ID NO: 913 gtcatggacttcagaaaat 1989 2008 SEQ ID NO:1916 atttgaaagaataaatgac 7028 7047 1 3 SEQ ID NO: 914tcaactctacaaatctgtt 2021 2040 SEQ ID NO: 1917 aacacattgaggctattga 69706989 1 3 SEQ ID NO: 915 aactctacaaatctgtttc 2023 2042 SEQ ID NO: 1918gaaaaaggggattgaagtt 10276 10295 1 3 SEQ ID NO: 916 aaatagaagggaatcttat2071 2090 SEQ ID NO: 1919 ataagcaaactgttaattt 5449 5468 1 3 SEQ ID NO:917 agaagggaatcttatattt 2075 2094 SEQ ID NO: 1920 aaatgcactgctgcgttct4892 4911 1 3 SEQ ID NO: 918 gaagggaatcttatatttg 2076 2095 SEQ ID NO:1921 caaaaacattttcaacttc 5279 5298 1 3 SEQ ID NO: 919tgatccaaataactacctt 2093 2112 SEQ ID NO: 1922 aaggaagaaagaaaaatca 34533472 1 3 SEQ ID NO: 920 tggatttgcttcagctgac 2150 2169 SEQ ID NO: 1923gtcagcccagttccttcca 10924 10943 1 3 SEQ ID NO: 921 tttgcttcagctgacctca2154 2173 SEQ ID NO: 1924 tgaggaaactcagatcaaa 12257 12276 1 3 SEQ ID NO:922 cttggaaggaaaaggcttt 2183 2202 SEQ ID NO: 1925 aaagcattggtagagcaag7842 7861 1 3 SEQ ID NO: 923 tggaaggaaaaggctttga 2185 2204 SEQ ID NO:1926 tcaagtctgtgggattcca 4078 4097 1 3 SEQ ID NO: 924ggctttgagccaacattgg 2196 2215 SEQ ID NO: 1927 ccaagaggtatttaaagcc 1295012969 1 3 SEQ ID NO: 925 tgagccaacattggaagct 2201 2220 SEQ ID NO: 1928agctttctgccactgctca 13513 13532 1 3 SEQ ID NO: 926 gagccaacattggaagctc2202 2221 SEQ ID NO: 1929 gagctttctgccactgctc 13512 13531 1 3 SEQ ID NO:927 aacattggaagctcttttt 2207 2226 SEQ ID NO: 1930 aaaagaaacagcatttgtt4531 4550 1 3 SEQ ID NO: 928 tggaagctctttttgggaa 2212 2231 SEQ ID NO:1931 ttccggcacgtgggttcca 3777 3796 1 3 SEQ ID NO: 929ctctttttgggaagcaagg 2218 2237 SEQ ID NO: 1932 ccttactgactttgcagag 77907809 1 3 SEQ ID NO: 930 tttttgggaagcaaggatt 2221 2240 SEQ ID NO: 1933aatcattgaaaaattaaaa 6722 6741 1 3 SEQ ID NO: 931 ttttcccagacagtgtcaa2239 2258 SEQ ID NO: 1934 ttgatgaaatcattgaaaa 6715 6734 1 3 SEQ ID NO:932 ttggctataccaaagatga 2323 2342 SEQ ID NO: 1935 tcattgctcccggagccaa2668 2687 1 3 SEQ ID NO: 933 ataccaaagatgataaaca 2329 2348 SEQ ID NO:1936 tgttgcttttgtaaagtat 6272 6291 1 3 SEQ ID NO: 934gagcaggatatggtaaatg 2349 2368 SEQ ID NO: 1937 catttcagccttcgggctc 42544273 1 3 SEQ ID NO: 935 atggtaaatggaataatgc 2358 2377 SEQ ID NO: 1938gcatgcctagtttctccat 9946 9965 1 3 SEQ ID NO: 936 tggtaaatggaataatgct2359 2378 SEQ ID NO: 1939 agcacagtacgaaaaacca 10801 10820 1 3 SEQ ID NO:937 taaatggaataatgctcag 2362 2381 SEQ ID NO: 1940 ctgaaagagatgaaattta13059 13078 1 3 SEQ ID NO: 938 tggaataatgctcagtgtt 2366 2385 SEQ ID NO:1941 aacagatttgaggattcca 7973 7992 1 3 SEQ ID NO: 939tcagtgttgagaagctgat 2377 2396 SEQ ID NO: 1942 atcacaactcctccactga 95349553 1 3 SEQ ID NO: 940 cagtgttgagaagctgatt 2378 2397 SEQ ID NO: 1943aatcacaactcctccactg 9533 9552 1 3 SEQ ID NO: 941 agtgttgagaagctgatta2379 2398 SEQ ID NO: 1944 taatcacaactcctccact 9532 9551 1 3 SEQ ID NO:942 gattaaagatttgaaatcc 2393 2412 SEQ ID NO: 1945 ggatactaagtaccaaatc6866 6885 1 3 SEQ ID NO: 943 gatttgaaatccaaagaag 2400 2419 SEQ ID NO:1946 cttccgtttaccagaaatc 8240 8259 1 3 SEQ ID NO: 944atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1947 acttccgtttaccagaaat 82398258 1 3 SEQ ID NO: 945 atccaaagaagtcccggaa 2408 2427 SEQ ID NO: 1948ttccaatttccctgtggat 3680 3699 1 3 SEQ ID NO: 946 tccaaagaagtcccggaag2409 2428 SEQ ID NO: 1949 cttccaatttccctgtgga 3679 3698 1 3 SEQ ID NO:947 agagcctacctccgcatct 2430 2449 SEQ ID NO: 1950 agattaatccgctggctct8563 8582 1 3 SEQ ID NO: 948 gagcctacctccgcatctt 2431 2450 SEQ ID NO:1951 aagattaatccgctggctc 8562 8581 1 3 SEQ ID NO: 949cttgggagaggagcttggt 2447 2466 SEQ ID NO: 1952 accactgggacctaccaag 1251912538 1 3 SEQ ID NO: 950 ggagcttggttttgccagt 2456 2475 SEQ ID NO: 1953actggtggcaaaaccctcc 2726 2745 1 3 SEQ ID NO: 951 ttggttttgccagtctcca2461 2480 SEQ ID NO: 1954 tggagaagccacactccaa 10763 10782 1 3 SEQ ID NO:952 cagtctccatgacctccag 2471 2490 SEQ ID NO: 1955 ctggtcgcctgccaaactg3530 3549 1 3 SEQ ID NO: 953 ctccatgacctccagctcc 2475 2494 SEQ ID NO:1956 ggagtcattgctcccggag 2664 2683 1 3 SEQ ID NO: 954ctgggaaagctgcttctga 2493 2512 SEQ ID NO: 1957 tcagaaagctaccttccag 79317950 1 3 SEQ ID NO: 955 gaggtcatcaggaagggct 2553 2572 SEQ ID NO: 1958agccagaagtgagatcctc 3506 3525 1 3 SEQ ID NO: 956 aagaatgacttttttcttc2574 2593 SEQ ID NO: 1959 gaaggcatctgggagtctt 3827 3846 1 3 SEQ ID NO:957 cttttttcttcactacatc 2582 2601 SEQ ID NO: 1960 gatgcttacaacactaaag6099 6118 1 3 SEQ ID NO: 958 catcttcatggagaatgcc 2597 2616 SEQ ID NO:1961 ggcacttccaaaattgatg 10710 10729 1 3 SEQ ID NO: 959cttcatggagaatgccttt 2600 2619 SEQ ID NO: 1962 aaagttaattgggaagaag 1227312292 1 3 SEQ ID NO: 960 aatgcctttgaactcccca 2610 2629 SEQ ID NO: 1963tgggctggcttcagccatt 5729 5748 1 3 SEQ ID NO: 961 gcctttgaactccccactg2613 2632 SEQ ID NO: 1964 cagtctgaacattgcaggc 5375 5394 1 3 SEQ ID NO:962 caaggctggagtaaaactg 2684 2703 SEQ ID NO: 1965 cagtgcaacgaccaacttg5072 5091 1 3 SEQ ID NO: 963 tggagtaaaactggaagta 2690 2709 SEQ ID NO:1966 tactccaacgccagctcca 3051 3070 1 3 SEQ ID NO: 964ggaagtagccaacatgcag 2702 2721 SEQ ID NO: 1967 ctgccatctcgagagttcc 40984117 1 3 SEQ ID NO: 965 tttgtgacaaatatgggca 2757 2776 SEQ ID NO: 1968tgcctttgtgtacaccaaa 11228 11247 1 3 SEQ ID NO: 966 tgtgacaaatatgggcatc2759 2778 SEQ ID NO: 1969 gatgggtctctacgccaca 4377 4396 1 3 SEQ ID NO:967 ggacttcgctaggagtggg 2786 2805 SEQ ID NO: 1970 cccaaggccacaggggtcc12333 12352 1 3 SEQ ID NO: 968 gtggggtccagatgaacac 2800 2819 SEQ ID NO:1971 gtgttctagacctctccac 4171 4190 1 3 SEQ ID NO: 969ttccacgagtcgggtctgg 2826 2845 SEQ ID NO: 1972 ccagaatctgtaccaggaa 1255412573 1 3 SEQ ID NO: 970 agtcgggtctggaggctca 2833 2852 SEQ ID NO: 1973tgagaactacgagctgact 4799 4818 1 3 SEQ ID NO: 971 tcgggtctggaggctcatg2835 2854 SEQ ID NO: 1974 catgaaggccaaattccga 7631 7650 1 3 SEQ ID NO:972 aaaagctgggaagctgaag 2861 2880 SEQ ID NO: 1975 cttccagacacctgatttt7943 7962 1 3 SEQ ID NO: 973 aagctgaagtttatcattc 2871 2890 SEQ ID NO:1976 gaatttacaattgttgctt 6261 6280 1 3 SEQ ID NO: 974gagaccagtcaagctgctc 2900 2919 SEQ ID NO: 1977 gagcttcaggaagcttctc 1320613225 1 3 SEQ ID NO: 975 gcaacacattacatttggt 2926 2945 SEQ ID NO: 1978accagtcagatattgttgc 10183 10202 1 3 SEQ ID NO: 976 acattacatttggtctcta2931 2950 SEQ ID NO: 1979 tagaatatgaactaaatgt 11881 11900 1 3 SEQ ID NO:977 cattacatttggtctctac 2932 2951 SEQ ID NO: 1980 gtagctgagaaaatcaatg7098 7117 1 3 SEQ ID NO: 978 aaacggaggtgatcccacc 2956 2975 SEQ ID NO:1981 ggtggataccctgaagttt 3197 3216 1 3 SEQ ID NO: 979attgagaacaggcagtcct 2979 2998 SEQ ID NO: 1982 aggaaaagcgcacctcaat 1202312042 1 3 SEQ ID NO: 980 tgagaacaggcagtcctgg 2981 3000 SEQ ID NO: 1983ccagcttccccacatctca 8333 8352 1 3 SEQ ID NO: 981 ctgcacctcaggcgcttac3035 3054 SEQ ID NO: 1984 gtaagaaaatacagagcag 6432 6451 1 3 SEQ ID NO:982 tccacagactccgcctcct 3066 3085 SEQ ID NO: 1985 aggacagagccttggtgga3184 3203 1 3 SEQ ID NO: 983 ctgaccggggacaccagat 3093 3112 SEQ ID NO:1986 atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 984tagagctggaactgaggcc 3112 3131 SEQ ID NO: 1987 ggcctctctggggcatcta 51365155 1 3 SEQ ID NO: 985 ctatgagctccagagagag 3167 3186 SEQ ID NO: 1988ctctcacaaaaaagtatag 6541 6560 1 3 SEQ ID NO: 986 cttggtggataccctgaag3194 3213 SEQ ID NO: 1989 cttcaggaagcttctcaag 13209 13228 1 3 SEQ ID NO:987 ttgtaactcaagcagaagg 3214 3233 SEQ ID NO: 1990 ccttacacaataatcacaa9522 9541 1 3 SEQ ID NO: 988 taactcaagcagaaggtgc 3217 3236 SEQ ID NO:1991 gcacctagctggaaagtta 6947 6966 1 3 SEQ ID NO: 989gcagaaggtgcgaagcaga 3225 3244 SEQ ID NO: 1992 tctgtgggattccatctgc 40834102 1 3 SEQ ID NO: 990 cagaaggtgcgaagcagac 3226 3245 SEQ ID NO: 1993gtctgtgggattccatctg 4082 4101 1 3 SEQ ID NO: 991 gtatgaccttgtccagtga3280 3299 SEQ ID NO: 1994 tcaccaacggagaacatac 10843 10862 1 3 SEQ ID NO:992 tatgaccttgtccagtgaa 3281 3300 SEQ ID NO: 1995 ttcaccaacggagaacata10842 10861 1 3 SEQ ID NO: 993 gaagtccaaattccggatt 3297 3316 SEQ ID NO:1996 aatctcaagctttctcttc 10044 10063 1 3 SEQ ID NO: 994gagggcaaaacgtcttaca 3363 3382 SEQ ID NO: 1997 tgtacaactggtccgcctc 42074226 1 3 SEQ ID NO: 995 agggcaaaacgtcttacag 3364 3383 SEQ ID NO: 1998ctgttaggacaccagccct 4054 4073 1 3 SEQ ID NO: 996 gactcaccctggacattca3382 3401 SEQ ID NO: 1999 tgaaattcaatcacaagtc 9068 9087 1 3 SEQ ID NO:997 ctggacattcagaacaaga 3390 3409 SEQ ID NO: 2000 tcttttcttttcagcccag9218 9237 1 3 SEQ ID NO: 998 tcatgggcgacctaagttg 3427 3446 SEQ ID NO:2001 caactgcagacatatatga 6627 6646 1 3 SEQ ID NO: 999tgggcgacctaagttgtga 3430 3449 SEQ ID NO: 2002 tcactccattaacctccca 63086327 1 3 SEQ ID NO: 1000 agttgtgacacaaaggaag 3441 3460 SEQ ID NO: 2003cttcttttccaattgaact 13830 13849 1 3 SEQ ID NO: 1001 tgacacaaaggaagaaaga3446 3465 SEQ ID NO: 2004 tcttcatcttcatctgtca 10212 10231 1 3 SEQ ID NO:1002 gacacaaaggaagaaagaa 3447 3466 SEQ ID NO: 2005 ttcttcatcttcatctgtc10211 10230 1 3 SEQ ID NO: 1003 ggaagaaagaaaaatcaag 3455 3474 SEQ ID NO:2006 cttgtcatgcctacgttcc 11340 11359 1 3 SEQ ID NO: 2007aaaaagcgatggccgggtc 3947 3966 SEQ ID NO: 2313 gaccttgcaagaatatttt 63356354 1 3 SEQ ID NO: 2008 gtcaaatataccttgaaca 3963 3982 SEQ ID NO: 2314tgttaacaaattccttgac 7355 7374 1 3 SEQ ID NO: 2009 tgaacaagaacagtttgaa3976 3995 SEQ ID NO: 2315 ttcaagttcctgaccttca 8302 8321 1 3 SEQ ID NO:2010 agtttgaaaattgagattc 3987 4006 SEQ ID NO: 2316 gaatctggctccctcaact9039 9058 1 3 SEQ ID NO: 2011 gtttgaaaattgagattcc 3988 4007 SEQ ID NO:2317 ggaaataccaagtcaaaac 10446 10465 1 3 SEQ ID NO: 2012ttgaaaattgagattcctt 3990 4009 SEQ ID NO: 2318 aaggaaaagcgcacctcaa 1202212041 1 3 SEQ ID NO: 2013 ctaaagatgttagagactg 4038 4057 SEQ ID NO: 2319cagttgaccacaagcttag 10537 10556 1 3 SEQ ID NO: 2014 atgttagagactgttagga4044 4063 SEQ ID NO: 2320 tccttaacaccttccacat 8065 8084 1 3 SEQ ID NO:2015 cagccctccacttcaagtc 4066 4085 SEQ ID NO: 2321 gacttctctagtcaggctg8805 8824 1 3 SEQ ID NO: 2016 agccctccacttcaagtct 4067 4086 SEQ ID NO:2322 agacatcgctgggctggct 5720 5739 1 3 SEQ ID NO: 2017ccatctgccatctcgagag 4094 4113 SEQ ID NO: 2323 ctctcaaatgacatgatgg 53225341 1 3 SEQ ID NO: 2018 attcccaagttgtatcaac 4134 4153 SEQ ID NO: 2324gttgagaagccccaagaat 6246 6265 1 3 SEQ ID NO: 2019 tcaactgcaagtgcctctc4148 4167 SEQ ID NO: 2325 gagatcaagacactgttga 8835 8854 1 3 SEQ ID NO:2020 ggtgttctagacctctcca 4170 4189 SEQ ID NO: 2326 tggaaccctctccctcacc4727 4746 1 3 SEQ ID NO: 2021 ctccacgaatgtctacagc 4184 4203 SEQ ID NO:2327 gctggtaacctaaaaggag 5580 5599 1 3 SEQ ID NO: 2022cacgaatgtctacagcaac 4187 4206 SEQ ID NO: 2328 gttgcccaccatcatcgtg 1166311682 1 3 SEQ ID NO: 2023 acgaatgtctacagcaact 4188 4207 SEQ ID NO: 2329agttgcccaccatcatcgt 11662 11681 1 3 SEQ ID NO: 2024 tcctacagtggtggcaaca4224 4243 SEQ ID NO: 2330 tgttagttgctcttaagga 13351 13370 1 3 SEQ ID NO:2025 cgttaccacatgaaggctg 4272 4291 SEQ ID NO: 2331 cagcaagtacctgagaacg8603 8622 1 3 SEQ ID NO: 2026 gaaggctgactctgtggtt 4283 4302 SEQ ID NO:2332 aacctatgccttaatcttc 13161 13180 1 3 SEQ ID NO: 2027tgtggttgacctgctttcc 4295 4314 SEQ ID NO: 2333 ggaaagttaaaacaacaca 69576976 1 3 SEQ ID NO: 2028 cctgctttcctacaatgtg 4304 4323 SEQ ID NO: 2334cacaccttgacattgcagg 11080 11099 1 3 SEQ ID NO: 2029 ctgctttcctacaatgtgc4305 4324 SEQ ID NO: 2335 gcacaccttgacattgcag 11079 11098 1 3 SEQ ID NO:2030 tcctacaatgtgcaaggat 4311 4330 SEQ ID NO: 2336 atccgctggctctgaagga8569 8588 1 3 SEQ ID NO: 2031 tatgaccacaagaatacgt 4344 4363 SEQ ID NO:2337 acgtccgtgtgccttcata 9976 9995 1 3 SEQ ID NO: 2032atgaccacaagaatacgtc 4345 4364 SEQ ID NO: 2338 gacgtccgtgtgccttcat 99759994 1 3 SEQ ID NO: 2033 gaatacgtctacactatca 4355 4374 SEQ ID NO: 2339tgattatctgaattcattc 6479 6498 1 3 SEQ ID NO: 2034 tttctagattcgaatatca4398 4417 SEQ ID NO: 2340 tgatttacatgatttgaaa 6677 6696 1 3 SEQ ID NO:2035 gattcgaatatcaaattca 4404 4423 SEQ ID NO: 2341 tgaagtagctgagaaaatc7094 7113 1 3 SEQ ID NO: 2036 gaaacaacccagtctcaaa 4441 4460 SEQ ID NO:2342 tttgaaaaattctcttttc 9206 9225 1 3 SEQ ID NO: 2037cccagtctcaaaaggttta 4448 4467 SEQ ID NO: 2343 taaattcattactcctggg 1129411313 1 3 SEQ ID NO: 2038 ctcaaaaggtttactaata 4454 4473 SEQ ID NO: 2344tattcaaaactgagttgag 12223 12242 1 3 SEQ ID NO: 2039 tcaaaaggtttactaatat4455 4474 SEQ ID NO: 2345 atattcaaaactgagttga 12222 12241 1 3 SEQ ID NO:2040 aaaaggtttactaatattc 4457 4476 SEQ ID NO: 2346 gaatttgaaagttcgtttt9272 9291 1 3 SEQ ID NO: 2041 gaaacagcatttgtttgtc 4535 4554 SEQ ID NO:2347 gacagcatcttcgtgtttc 11206 11225 1 3 SEQ ID NO: 2042atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 2348 acttaaaaaatataaaaat 80148033 1 3 SEQ ID NO: 2043 tcaagattgatgggcagtt 4561 4580 SEQ ID NO: 2349aactctcaagtcaagttga 13414 13433 1 3 SEQ ID NO: 2044 ttcagagtctcttcgttct4578 4597 SEQ ID NO: 2350 agaagatggcaaatttgaa 11987 12006 1 3 SEQ ID NO:2045 cagagtctcttcgttctat 4580 4599 SEQ ID NO: 2351 atagcatggacttcttctg8865 8884 1 3 SEQ ID NO: 2046 atgctaaaggcacatatgg 4597 4616 SEQ ID NO:2352 ccatttgagatcacggcat 9237 9256 1 3 SEQ ID NO: 2047gcacatatggcctgtcttg 4606 4625 SEQ ID NO: 2353 caagttggcaagtaagtgc 93649383 1 3 SEQ ID NO: 2048 gagtccaacctgaggttta 4659 4678 SEQ ID NO: 2354taaagtgccacttttactc 6182 6201 1 3 SEQ ID NO: 2049 agtccaacctgaggtttaa4660 4679 SEQ ID NO: 2355 ttaacagggaagatagact 9300 9319 1 3 SEQ ID NO:2050 cctacctccaaggcaccaa 4684 4703 SEQ ID NO: 2356 ttggcaagtaagtgctagg9368 9387 1 3 SEQ ID NO: 2051 gaagatggaaccctctccc 4722 4741 SEQ ID NO:2357 gggaagaagaggcagcttc 12283 12302 1 3 SEQ ID NO: 2052tgatctgcaaagtggcatc 4754 4773 SEQ ID NO: 2358 gatgaggaaactcagatca 1225512274 1 3 SEQ ID NO: 2053 gatctgcaaagtggcatca 4755 4774 SEQ ID NO: 2359tgatgaggaaactcagatc 12254 12273 1 3 SEQ ID NO: 2054 gcttccctaaagtatgaga4785 4804 SEQ ID NO: 2360 tctcgtgtctaggaaaagc 5969 5988 1 3 SEQ ID NO:2055 gtatgagaactacgagctg 4796 4815 SEQ ID NO: 2361 cagcttaagagacacatac6912 6931 1 3 SEQ ID NO: 2056 tctaacaagatggatatga 4860 4879 SEQ ID NO:2362 tcattttccaactaataga 13024 13043 1 3 SEQ ID NO: 2057ctgctgcgttctgaatatc 4899 4918 SEQ ID NO: 2363 gatacaagaaaaactgcag 68936912 1 3 SEQ ID NO: 2058 tcattgaggttcttcagcc 4932 4951 SEQ ID NO: 2364ggctcatatgctgaaatga 5340 5359 1 3 SEQ ID NO: 2059 ttctggatcactaaattcc4955 4974 SEQ ID NO: 2365 ggaaggacaaggcccagaa 12541 12560 1 3 SEQ ID NO:2060 ccatggtcttgagttaaat 4973 4992 SEQ ID NO: 2366 atttttattcctgccatgg10095 10114 1 3 SEQ ID NO: 2061 tcttaggcactgacaaaat 4999 5018 SEQ ID NO:2367 attttttgcaagttaaaga 14011 14030 1 3 SEQ ID NO: 2062acaaggcgacactaaggat 5032 5051 SEQ ID NO: 2368 atccatgatctacatttgt 67866805 1 3 SEQ ID NO: 2063 tgcaacgaccaacttgaag 5075 5094 SEQ ID NO: 2369cttcagggaacacaatgca 5177 5196 1 3 SEQ ID NO: 2064 caacttgaagtgtagtctc5084 5103 SEQ ID NO: 2370 gagatgagagatgccgttg 6231 6250 1 3 SEQ ID NO:2065 gctggagaatgagctgaat 5108 5127 SEQ ID NO: 2371 attctcttttcttttcagc9214 9233 1 3 SEQ ID NO: 2066 gcagagcttggcctctctg 5127 5146 SEQ ID NO:2372 cagatacaagaaaaactgc 6891 6910 1 3 SEQ ID NO: 2067tctctggggcatctatgaa 5140 5159 SEQ ID NO: 2373 ttcattcaattgggagaga 64916510 1 3 SEQ ID NO: 2068 tctggggcatctatgaaat 5142 5161 SEQ ID NO: 2374atttgtaagaaaatacaga 6428 6447 1 3 SEQ ID NO: 2069 aacacaatgcaaaattcag5185 5204 SEQ ID NO: 2375 ctgaagcattaaaactgtt 7498 7517 1 3 SEQ ID NO:2070 ctcacagagctatcactgg 5223 5242 SEQ ID NO: 2376 ccagatgctgaacagtgag8141 8160 1 3 SEQ ID NO: 2071 tgggaagtgcttatcaggc 5239 5258 SEQ ID NO:2377 gcctacgttccatgtccca 11348 11367 1 3 SEQ ID NO: 2072ttcaaggtcagtcaagaag 5295 5314 SEQ ID NO: 2378 cttcagtgcagaatatgaa 1196911988 1 3 SEQ ID NO: 2073 aatgacatgatgggctcat 5328 5347 SEQ ID NO: 2379atgattatctgaattcatt 6478 6497 1 3 SEQ ID NO: 2074 gctcatatgctgaaatgaa5341 5360 SEQ ID NO: 2380 ttcagccattgacatgagc 5738 5757 1 3 SEQ ID NO:2075 atatgctgaaatgaaattt 5345 5364 SEQ ID NO: 2381 aaatagctattgctaatat6694 6713 1 3 SEQ ID NO: 2076 tctgaacattgcaggctta 5378 5397 SEQ ID NO:2382 taagaaccagaagatcaga 10988 11007 1 3 SEQ ID NO: 2077gaacattgcaggcttatca 5381 5400 SEQ ID NO: 2383 tgatatcgacgtgaggttc 1248212501 1 3 SEQ ID NO: 2078 tgcaggcttatcactggac 5387 5406 SEQ ID NO: 2384gtcctggattccacatgca 11844 11863 1 3 SEQ ID NO: 2079 tcaaaacttgacaacattt5412 5431 SEQ ID NO: 2385 aaattccttgacatgttga 7362 7381 1 3 SEQ ID NO:2080 atttacagctctgacaagt 5427 5446 SEQ ID NO: 2386 acttaaaaaatataaaaat8014 8033 1 3 SEQ ID NO: 2081 ctctgacaagttttataag 5435 5454 SEQ ID NO:2387 cttacttgaattccaagag 10666 10685 1 3 SEQ ID NO: 2082gttaatttacagctacagc 5460 5479 SEQ ID NO: 2388 gctgcatgtggctggtaac 55705589 1 3 SEQ ID NO: 2083 ttctctggtaactacttta 5483 5502 SEQ ID NO: 2389taaaagattactttgagaa 7267 7286 1 3 SEQ ID NO: 2084 cctaaaaggagcctaccaa5588 5507 SEQ ID NO: 2390 ttggcaagtaagtgctagg 9368 9387 1 3 SEQ ID NO:2085 aaaaggagcctaccaaaat 5591 5610 SEQ ID NO: 2391 atttacaattgttgctttt6263 6282 1 3 SEQ ID NO: 2086 aggagcctaccaaaataat 5594 5613 SEQ ID NO:2392 attacctatgatttctcct 10119 10138 1 3 SEQ ID NO: 2087ataatgaaataaaacacat 5608 5627 SEQ ID NO: 2393 atgtcaaacactttgttat 70577076 1 3 SEQ ID NO: 2088 aaaacacatctatgccatc 5618 5637 SEQ ID NO: 2394gatgaagatgacgactttt 12150 12169 1 3 SEQ ID NO: 2089 tgctaaggttcagggtgtg5678 5697 SEQ ID NO: 2395 cacaagtcgattcccagca 9079 9098 1 3 SEQ ID NO:2090 gagtttagccatcggctca 5697 5716 SEQ ID NO: 2396 tgaggtgactcagagactc7442 7461 1 3 SEQ ID NO: 2091 gctggcttcagccattgac 5732 5751 SEQ ID NO:2397 gtcagtgaagttctccagc 8588 8607 1 3 SEQ ID NO: 2092atttcagcaatgtcttccg 5782 5801 SEQ ID NO: 2398 cggagcatgggagtgaaat 86208639 1 3 SEQ ID NO: 2093 tttcagcaatgtcttccgt 5783 5802 SEQ ID NO: 2399acggagcatgggagtgaaa 8619 8638 1 3 SEQ ID NO: 2094 ttcagcaatgtcttccgtt5784 5803 SEQ ID NO: 2400 aacggagcatgggagtgaa 8618 8637 1 3 SEQ ID NO:2095 cagcaatgtcttccgttct 5786 5805 SEQ ID NO: 2401 agaagtgtcttcaaagctg12404 12423 1 3 SEQ ID NO: 2096 tgtcttccgttctgtaatg 5792 5811 SEQ ID NO:2402 cattcaattgggagagaca 6493 6512 1 3 SEQ ID NO: 2097gtcttccgttctgtaatgg 5793 5812 SEQ ID NO: 2403 ccattcagtctctcaagac 1296712986 1 3 SEQ ID NO: 2098 atgggaaactcgctctctg 5851 5870 SEQ ID NO: 2404cagataaaaaactcaccat 12205 12224 1 3 SEQ ID NO: 2099 ggagaacatactgggcagc5871 5890 SEQ ID NO: 2405 gctgttttgaagactctcc 1080 1099 1 3 SEQ ID NO:2100 gttgaaagcagaacctctg 5906 5925 SEQ ID NO: 2406 cagaattcataatcccaac8266 8285 1 3 SEQ ID NO: 2101 gtctaggaaaagcatcagt 5975 5994 SEQ ID NO:2407 actgcaagatttttcagac 13604 13623 1 3 SEQ ID NO: 2102agcatcagtgcagctcttg 5985 6004 SEQ ID NO: 2408 caagaacctgttagttgct 1334313362 1 3 SEQ ID NO: 2103 ttgaacacaaagtcagtgc 6001 6020 SEQ ID NO: 2409gcacatcaatattgatcaa 6410 6429 1 3 SEQ ID NO: 2104 gcagacaggcacctggaaa6038 6057 SEQ ID NO: 2410 tttcagatggcattgctgc 11602 11621 1 3 SEQ ID NO:2105 gaaactcaagacccaattt 6053 6072 SEQ ID NO: 2411 aaatcccatccaggttttc8029 8048 1 3 SEQ ID NO: 2106 acaatgaatacagccagga 6076 6095 SEQ ID NO:2412 tcctttggctgtgctttgt 9674 9693 1 3 SEQ ID NO: 2107cttggatgcttacaacact 6095 6114 SEQ ID NO: 2413 agtgaagttctccagcaag 85918610 1 3 SEQ ID NO: 2108 ttggcgtggagcttactgg 6124 6143 SEQ ID NO: 2414ccagaattcataatcccaa 8265 8284 1 3 SEQ ID NO: 2109 cacttttactcagtgagcc6190 6209 SEQ ID NO: 2415 ggctattgatgttagagtg 6980 6999 1 3 SEQ ID NO:2110 tttagagatgagagatgcc 6227 6246 SEQ ID NO: 2416 ggcatgatgctcatttaaa9169 9188 1 3 SEQ ID NO: 2111 gagaagccccaagaattta 6249 6268 SEQ ID NO:2417 taaagccattcagtctctc 12962 12981 1 3 SEQ ID NO: 2112caattgttgcttttgtaaa 6268 6287 SEQ ID NO: 2418 tttaaccagtcagatattg 1017910198 1 3 SEQ ID NO: 2113 ttttgtaaagtatgataaa 6278 6297 SEQ ID NO: 2419tttattgctgaatccaaaa 13647 13666 1 3 SEQ ID NO: 2114 ttgtaaagtatgataaaaa6280 6299 SEQ ID NO: 2420 ttttgagaggaatcgacaa 6350 6369 1 3 SEQ ID NO:2115 ttcactccattaacctccc 6307 6326 SEQ ID NO: 2421 gggaaaaaacaggcttgaa9568 9587 1 3 SEQ ID NO: 2116 ttttgagaccttgcaagaa 6329 6348 SEQ ID NO:2422 ttctctctatgggaaaaaa 9558 9577 1 3 SEQ ID NO: 2117accttgcaagaatattttg 6336 6355 SEQ ID NO: 2423 caaaagaagcccaagaggt 1294012959 1 3 SEQ ID NO: 2118 tcaatattgatcaatttgt 6415 6434 SEQ ID NO: 2424acaaagcagattatgttga 11821 11840 1 3 SEQ ID NO: 2119 cagagcagccctgggaaaa6443 6462 SEQ ID NO: 2425 ttttcagaccaactctctg 13614 13633 1 3 SEQ ID NO:2120 cctgggaaaactcccacag 6452 6471 SEQ ID NO: 2426 ctgtctctggtcagccagg7716 7735 1 3 SEQ ID NO: 2121 actcccacagcaagctaat 6461 6480 SEQ ID NO:2427 attacacttcctttcgagt 12861 12880 1 3 SEQ ID NO: 2122aattcattcaattgggaga 6489 6508 SEQ ID NO: 2428 tctcttcctccatggaatt 1047110490 1 3 SEQ ID NO: 2123 ttcaattgggagagacaag 6495 6514 SEQ ID NO: 2429cttggagtgccagtttgaa 11800 11819 1 3 SEQ ID NO: 2124 aggagaaactgactgctct6526 6545 SEQ ID NO: 2430 agagcttatgggatttcct 11155 11174 1 3 SEQ ID NO:2125 actgactgctctcacaaaa 6533 6552 SEQ ID NO: 2431 ttttggcaagctatacagt8372 8391 1 3 SEQ ID NO: 2126 gactgctctcacaaaaaag 6536 6555 SEQ ID NO:2432 ctttgtgagtttatcagtc 9687 9706 1 3 SEQ ID NO: 2127cagacatatatgatacaat 6633 6652 SEQ ID NO: 2433 attggatatccaagatctg 19251944 1 3 SEQ ID NO: 2128 aatttgatcagtatattaa 6849 6668 SEQ ID NO: 2434ttaaaagaaatcttcaatt 13807 13826 1 3 SEQ ID NO: 2129 tatgatttacatgatttga6675 6694 SEQ ID NO: 2435 tcaatgattatatcccata 13120 13139 1 3 SEQ ID NO:2130 tttgaaaatagctattgct 6689 6708 SEQ ID NO: 2436 agcacagaaaaaattcaaa13856 13875 1 3 SEQ ID NO: 2131 ttgaaaatagctattgcta 6690 6709 SEQ ID NO:2437 tagcacagaaaaaattcaa 13855 13874 1 3 SEQ ID NO: 2132aatagctattgctaatatt 6695 6714 SEQ ID NO: 2438 aataaatggagtctttatt 1407614095 1 3 SEQ ID NO: 2133 attattgatgaaatcattg 6711 6730 SEQ ID NO: 2439caataccagaattcataat 8260 8279 1 3 SEQ ID NO: 2134 aaagtcttgatgagcacta6739 6758 SEQ ID NO: 2440 tagtgattacacttccttt 12856 12875 1 3 SEQ ID NO:2135 aagtcttgatgagcactat 6740 6759 SEQ ID NO: 2441 atagcaacactaaatactt8761 8780 1 3 SEQ ID NO: 2136 ttgatgagcactatcatat 6745 6764 SEQ ID NO:2442 atatccaagatgagatcaa 13093 13112 1 3 SEQ ID NO: 2137taattttagtaaaaacaat 6769 6788 SEQ ID NO: 2443 attgagattccctccatta 1169411713 1 3 SEQ ID NO: 2138 ttttagtaaaaacaatcca 6772 6791 SEQ ID NO: 2444tggagtgccagtttgaaaa 11802 11821 1 3 SEQ ID NO: 2139 acatttgtttattgaaaat6797 6816 SEQ ID NO: 2445 atttcctaaagctggatgt 11167 11186 1 3 SEQ ID NO:2140 attgattttaacaaaagtg 6816 6835 SEQ ID NO: 2446 cactgttccagttgtcaat9863 9882 1 3 SEQ ID NO: 2141 attttaacaaaagtggaag 6820 6839 SEQ ID NO:2447 cttaaaagacttaaaaaat 8006 8025 1 3 SEQ ID NO: 2142aaatcagaatccagataca 6880 6899 SEQ ID NO: 2448 tgtaccataagccatattt 1008010099 1 3 SEQ ID NO: 2143 gaatccagatacaagaaaa 6886 6905 SEQ ID NO: 2449ttttctaaacttgaaattc 9057 9076 1 3 SEQ ID NO: 2144 ttaagagacacatacagaa6916 6935 SEQ ID NO: 2450 ttcttaaacattcctttaa 9483 9502 1 3 SEQ ID NO:2145 atccagcacctagctggaa 6942 6961 SEQ ID NO: 2451 ttccaatttccctgtggat3680 3699 1 3 SEQ ID NO: 2146 tgagcatgtcaaacacttt 7052 7071 SEQ ID NO:2452 aaagtgccacttttactca 6183 6202 1 3 SEQ ID NO: 2147gagcatgtcaaacactttg 7053 7072 SEQ ID NO: 2453 caaatgacatgatgggctc 53265345 1 3 SEQ ID NO: 2148 aaacactttgttataaatc 7062 7081 SEQ ID NO: 2454gattatatcccatatgttt 13125 13144 1 3 SEQ ID NO: 2149 tgagaaaatcaatgccttc7103 7122 SEQ ID NO: 2455 gaaggaaaagcgcacctca 12021 12040 1 3 SEQ ID NO:2150 tatgaagtagaccaacaaa 7152 7171 SEQ ID NO: 2456 tttgtggagggtagtcata10323 10342 1 3 SEQ ID NO: 2151 aagtagaccaacaaatcca 7156 7175 SEQ ID NO:2457 tggatgaagatgacgactt 12148 12167 1 3 SEQ ID NO: 2152aagttgaaggagactattc 7215 7234 SEQ ID NO: 2458 gaataccaatgctgaactt 1016010179 1 3 SEQ ID NO: 2153 acaagttaagataaaagat 7256 7275 SEQ ID NO: 2459atctaaattcagttcttgt 11326 11345 1 3 SEQ ID NO: 2154 aagataaaagattactttg7263 7282 SEQ ID NO: 2460 caaaatagaagggaatctt 2069 2088 1 3 SEQ ID NO:2155 gattactttgagaaattag 7272 7291 SEQ ID NO: 2461 ctaaacttgaaattcaatc9061 9080 1 3 SEQ ID NO: 2156 tgagaaattagttggattt 7280 7299 SEQ ID NO:2462 aaatccgtgaggtgactca 7435 7454 1 3 SEQ ID NO: 2157aaattagttggatttattg 7284 7303 SEQ ID NO: 2463 caattttgagaatgaattt 1041110430 1 3 SEQ ID NO: 2158 tggatttattgatgatgct 7292 7311 SEQ ID NO: 2464agcatgcctagtttctcca 9945 9964 1 3 SEQ ID NO: 2159 tcattgaagatgttaacaa7345 7364 SEQ ID NO: 2465 ttgtagatgaaaccaatga 7414 7433 1 3 SEQ ID NO:2160 cattgaagatgttaacaaa 7346 7365 SEQ ID NO: 2466 tttgtagatgaaaccaatg7413 7432 1 3 SEQ ID NO: 2161 attgaagatgttaacaaat 7347 7366 SEQ ID NO:2467 atttaagtatgatttcaat 10487 10506 1 3 SEQ ID NO: 2162ttgaagatgttaacaaatt 7348 7367 SEQ ID NO: 2468 aatttaagtatgatttcaa 1048610505 1 3 SEQ ID NO: 2163 tgaagatgttaacaaattc 7349 7368 SEQ ID NO: 2469gaatttaagtatgatttca 10485 10504 1 3 SEQ ID NO: 2164 acatgttgataaagaaatt7372 7391 SEQ ID NO: 2470 aattccctgaagttgatgt 11479 11498 1 3 SEQ ID NO:2165 tttgattaccaccagtttg 7398 7417 SEQ ID NO: 2471 caaattgaacatccccaaa8783 8802 1 3 SEQ ID NO: 2166 caaaatccgtgaggtgact 7433 7452 SEQ ID NO:2472 agtccccctaacagatttg 7964 7983 1 3 SEQ ID NO: 2167aaaatccgtgaggtgactc 7434 7453 SEQ ID NO: 2473 gagtgaaatgctgtttttt 86308649 1 3 SEQ ID NO: 2168 aggtgactcagagactcaa 7444 7463 SEQ ID NO: 2474ttgatgatatctggaacct 10723 10742 1 3 SEQ ID NO: 2169 gtgaaattcaggctctgga7465 7484 SEQ ID NO: 2475 tccaatctcctcttttcac 8401 8420 1 3 SEQ ID NO:2170 gttgcagtgtatctggaaa 7539 7558 SEQ ID NO: 2476 tttcaagcaaatgcacaac8532 8551 1 3 SEQ ID NO: 2171 ttaagttcagcatctttgg 7608 7627 SEQ ID NO:2477 ccaatgctgaactttttaa 10165 10184 1 3 SEQ ID NO: 2172tgaaggccaaattccgaga 7633 7852 SEQ ID NO: 2478 tctcctttcttcatcttca 1020510224 1 3 SEQ ID NO: 2173 aatgtatcaaatggacatt 7676 7695 SEQ ID NO: 2479aatgaagtccggattcatt 11013 11032 1 3 SEQ ID NO: 2174 attcagcaggaacttcaac7692 7711 SEQ ID NO: 2480 gttgagaagccccaagaat 6246 6265 1 3 SEQ ID NO:2175 acctgtctctggtcagcca 7714 7733 SEQ ID NO: 2481 tggcaagtaagtgctaggt9369 9388 1 3 SEQ ID NO: 2176 cctgtctctggtcagccag 7715 7734 SEQ ID NO:2482 ctggacttctctagtcagg 8802 8821 1 3 SEQ ID NO: 2177ggtcagccaggtttatagc 7724 7743 SEQ ID NO: 2483 gctaaaggagcagttgacc 1052710546 1 3 SEQ ID NO: 2178 ccaggtttatagcacactt 7730 7749 SEQ ID NO: 2484aagtccggattcattctgg 11017 11036 1 3 SEQ ID NO: 2179 gtttatagcacacttgtca7734 7753 SEQ ID NO: 2485 tgacctgtccattcaaaac 13673 13692 1 3 SEQ ID NO:2180 acttgtcacctacatttct 7745 7764 SEQ ID NO: 2486 agaaaaaggggattgaagt10275 10294 1 3 SEQ ID NO: 2181 ctgattggtggactcttgc 7762 7781 SEQ ID NO:2487 gcaagttaaagaaaatcag 14018 14037 1 3 SEQ ID NO: 2182atgaaagcattggtagagc 7839 7858 SEQ ID NO: 2488 gctcatctcctttcttcat 1020010219 1 3 SEQ ID NO: 2183 tgaaagcattggtagagca 7840 7859 SEQ ID NO: 2489tgctcatctcctttcttca 10199 10218 1 3 SEQ ID NO: 2184 gggttcactgttcctgaaa7860 7879 SEQ ID NO: 2490 tttcaccatagaaggaccc 8951 8970 1 3 SEQ ID NO:2185 tcaagaccatccttgggac 7879 7898 SEQ ID NO: 2491 gtccccctaacagatttga7965 7984 1 3 SEQ ID NO: 2186 ccttgggaccatgcctgcc 7889 7908 SEQ ID NO:2492 ggcaccagggctcggaagg 13970 13989 1 3 SEQ ID NO: 2187ttcaggctcttcagaaagc 7921 7940 SEQ ID NO: 2493 gcttgaaggaattcttgaa 95809599 1 3 SEQ ID NO: 2188 ttcagataaacttcaaaga 7996 8015 SEQ ID NO: 2494tcttcataagttcaatgaa 13175 13194 1 3 SEQ ID NO: 2189 acttcaaagacttaaaaaa8005 8024 SEQ ID NO: 2495 ttttaacaaaagtggaagt 6821 6840 1 3 SEQ ID NO:2190 atcccatccaggttttcca 8031 8050 SEQ ID NO: 2496 tggagaagcaaatctggat9464 9483 1 3 SEQ ID NO: 2191 gaatttaccatccttaaca 8055 8074 SEQ ID NO:2497 tgttgaagtgtctccattc 9881 9900 1 3 SEQ ID NO: 2192cattccttcctttacaatt 8081 8100 SEQ ID NO: 2498 aattccaattttgagaatg 1040610425 1 3 SEQ ID NO: 2193 ttgaccagatgctgaacag 8137 8156 SEQ ID NO: 2499ctgttgaaagatttatcaa 12924 12943 1 3 SEQ ID NO: 2194 aatcaccctgccagacttc8225 8244 SEQ ID NO: 2500 gaagttctcaattttgatt 8514 8533 1 3 SEQ ID NO:2195 tgaccttcacataccagaa 8312 8331 SEQ ID NO: 2501 ttcttctggaaaagggtca8876 8895 1 3 SEQ ID NO: 2196 ttccagcttccccacatct 8331 8350 SEQ ID NO:2502 agattctcagatgagggaa 8913 8932 1 3 SEQ ID NO: 2197aagctatacagtattctga 8379 8398 SEQ ID NO: 2503 tcagatggcattgctgctt 1160411623 1 3 SEQ ID NO: 2198 attctgaaaatccaatctc 8391 8410 SEQ ID NO: 2504gagataaccgtgcctgaat 11544 11563 1 3 SEQ ID NO: 2199 tttcacattagatgcaaat8414 8433 SEQ ID NO: 2505 attttgaaaaaaacagaaa 9730 9749 1 3 SEQ ID NO:2200 caaatgctgacatagggaa 8428 8447 SEQ ID NO: 2506 ttccatcacaaatcctttg9662 9681 1 3 SEQ ID NO: 2201 gagagtccaaattagaagt 8500 8519 SEQ ID NO:2507 actttacttcccaactctc 13402 13421 1 3 SEQ ID NO: 2202agagtccaaattagaagtt 8501 8520 SEQ ID NO: 2508 aactttacttcccaactct 1340113420 1 3 SEQ ID NO: 2203 tctcaattttgattttcaa 8519 8538 SEQ ID NO: 2509ttgattcccttttttgaga 11529 11548 1 3 SEQ ID NO: 2204 caattttgattttcaagca8522 8541 SEQ ID NO: 2510 tgctgaatccaaaagattg 13652 13671 1 3 SEQ ID NO:2205 aatgcacaactctcaaacc 8541 8560 SEQ ID NO: 2511 ggtttatcaaggggccatt12452 12471 1 3 SEQ ID NO: 2206 agttctccagcaagtacct 8596 8615 SEQ ID NO:2512 aggttccatcgtgcaaact 11380 11399 1 3 SEQ ID NO: 2207agtacctgagaacggagca 8608 8627 SEQ ID NO: 2513 tgctccaggagaacttact 1377213791 1 3 SEQ ID NO: 2208 tcaaacacagtggcaagtt 8670 8689 SEQ ID NO: 2514aactctcaagtcaagttga 13414 13433 1 3 SEQ ID NO: 2209 acaatcagcttaccctgga8743 8762 SEQ ID NO: 2515 tccattctgaatatattgt 13372 13391 1 3 SEQ ID NO:2210 ctggatagcaacactaaat 8757 8776 SEQ ID NO: 2516 attttctgaacttccccag12694 12713 1 3 SEQ ID NO: 2211 ctgacctgcgcaacgagat 8821 8840 SEQ ID NO:2517 atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 2212agatgagggaacacatgaa 8921 8940 SEQ ID NO: 2518 ttcatgtccctagaaatct 1003010049 1 3 SEQ ID NO: 2213 tcaacttttctaaacttga 9052 9071 SEQ ID NO: 2519tcaaggataacgtgtttga 12610 12629 1 3 SEQ ID NO: 2214 ttctaaacttgaaattcaa9059 9078 SEQ ID NO: 2520 ttgatgatgctgtcaagaa 7300 7319 1 3 SEQ ID NO:2215 gaaattcaatcacaagtcg 9069 9088 SEQ ID NO: 2521 cgacgaagaaaataatttc13558 13577 1 3 SEQ ID NO: 2216 cactgtttggagaagggaa 9133 9152 SEQ ID NO:2522 ttccagaaagcagccagtg 12498 12517 1 3 SEQ ID NO: 2217actgtttggagaagggaag 9134 9153 SEQ ID NO: 2523 cttccccaaagagaccagt 28902909 1 3 SEQ ID NO: 2218 aattctcttttcttttcag 9213 9232 SEQ ID NO: 2524ctgattactatgaaaaatt 13630 13649 1 3 SEQ ID NO: 2219 ttcttttcagcccagccat9222 9241 SEQ ID NO: 2525 atggaaaagggaaagagaa 13486 13505 1 3 SEQ ID NO:2220 tttgaaagttcgttttcca 9275 9294 SEQ ID NO: 2526 tggaagtgtcagtggcaaa10372 10391 1 3 SEQ ID NO: 2221 cagggaagataggcttcct 9304 9323 SEQ ID NO:2527 aggacctttcaaattcctg 9840 9859 1 3 SEQ ID NO: 2222ataagtacaaccaaaattt 9397 9416 SEQ ID NO: 2528 aaatcaggatctgagttat 1403014049 1 3 SEQ ID NO: 2223 acaacgagaacattatgga 9427 9446 SEQ ID NO: 2529tccattctgaatatattgt 13372 13391 1 3 SEQ ID NO: 2224 aggaataaatggagaagca9455 9474 SEQ ID NO: 2530 tgctggaattgtcattcct 11726 11745 1 3 SEQ ID NO:2225 agcaaatctggatttctta 9470 9489 SEQ ID NO: 2531 taagttctctgtacctgct11711 11730 1 3 SEQ ID NO: 2226 tcctttaacaattcctgaa 9494 9513 SEQ ID NO:2532 ttcaaaacgagcttcagga 13198 13217 1 3 SEQ ID NO: 2227tttaacaattcctgaaatg 9497 9516 SEQ ID NO: 2533 catttgatttaagtgtaaa 96139632 1 3 SEQ ID NO: 2228 acacaataatcacaactcc 9526 9545 SEQ ID NO: 2534ggagacagcatcttcgtgt 11203 11222 1 3 SEQ ID NO: 2229 aagatttctctctatggga9553 9572 SEQ ID NO: 2535 tcccagaaaacctcttctt 3928 3947 1 3 SEQ ID NO:2230 gaaaaaacaggcttgaagg 9570 9589 SEQ ID NO: 2536 ccttttacaattcattttc13013 13032 1 3 SEQ ID NO: 2231 ttgaaggaattcttgaaaa 9582 9601 SEQ ID NO:2537 ttttgagaatgaatttcaa 10414 10433 1 3 SEQ ID NO: 2232tgaaggaattcttgaaaac 9583 9602 SEQ ID NO: 2538 gttttggctgataaattca 1128311302 1 3 SEQ ID NO: 2233 agctcagtataagaaaaac 9632 9651 SEQ ID NO: 2539gtttgataagtacaaagct 9797 9816 1 3 SEQ ID NO: 2234 tcaaatcctttgacaggca9712 9731 SEQ ID NO: 2540 tgcctgagcagaccattga 11680 11699 1 3 SEQ ID NO:2235 atgaaacaaaaattaagtt 9781 9800 SEQ ID NO: 2541 aactttgcactatgttcat12754 12773 1 3 SEQ ID NO: 2236 aattcctggatacactgtt 9851 9870 SEQ ID NO:2542 aacacatgaatcacaaatt 8930 8949 1 3 SEQ ID NO: 2237ttccagttgtcaatgttga 9868 9887 SEQ ID NO: 2543 tcaaaacgagcttcaggaa 1319913218 1 3 SEQ ID NO: 2238 aagtgtctccattcaccat 9886 9905 SEQ ID NO: 2544atgggaagtataagaactt 4834 4853 1 3 SEQ ID NO: 2239 gtcagcatgcctagtttct9942 9961 SEQ ID NO: 2545 agaaaaggcacaccttgac 11072 11091 1 3 SEQ ID NO:2240 ctgccatgggcaatattac 10105 10124 SEQ ID NO: 2546 gtaagaaaatacagagcag6432 6451 1 3 SEQ ID NO: 2241 tgaataccaatgctgaact 10159 10178 SEQ ID NO:2547 agttgaaggagactattca 7216 7235 1 3 SEQ ID NO: 2242tattgttgctcatctcctt 10193 10212 SEQ ID NO: 2548 aaggaaacataaactaata12881 12900 1 3 SEQ ID NO: 2243 tgttgctcatctcctttct 10196 10215 SEQ IDNO: 2549 agaagaaatctgcagaaca 12423 12442 1 3 SEQ ID NO: 2244tctgtcattgatgcactgc 10224 10243 SEQ ID NO: 2550 gcagtagactataagcaga13920 13939 1 3 SEQ ID NO: 2245 ccacagctctgtctctgag 10297 10316 SEQ IDNO: 2551 ctcagggatctgaaggtgg 8187 8206 1 3 SEQ ID NO: 2246atttgtggagggtagtcat 10322 10341 SEQ ID NO: 2552 atgaagtagaccaacaaat 71537172 1 3 SEQ ID NO: 2247 atatggaagtgtcagtggc 10369 10388 SEQ ID NO: 2553gccacactccaacgcatat 10770 10789 1 3 SEQ ID NO: 2248 tggaaataccaagtcaaaa10445 10464 SEQ ID NO: 2554 ttttacaattcattttcca 13015 13034 1 3 SEQ IDNO: 2249 aagtcaaaacctactgtct 10455 10474 SEQ ID NO: 2555agacctagtgattacactt 12851 12870 1 3 SEQ ID NO: 2250 actgtctcttcctccatgg10467 10486 SEQ ID NO: 2556 ccatgcaagtcagcccagt 10916 10935 1 3 SEQ IDNO: 2251 cttcctccatggaatttaa 10474 10493 SEQ ID NO: 2557ttaatcgagaggtatgaag 7140 7159 1 3 SEQ ID NO: 2252 attcttcaatgctgtactc10504 10523 SEQ ID NO: 2558 gagttgagggtccgggaat 12234 12253 1 3 SEQ IDNO: 2253 ttgaccacaagcftagctt 10540 10559 SEQ ID NO: 2559aagcgcacctcaatatcaa 12028 12047 1 3 SEQ ID NO: 2254 cctcacctcttacttttcc10565 10584 SEQ ID NO: 2560 ggaactattgctagtgagg 10641 10660 1 3 SEQ IDNO: 2255 agctgcagggcacttccaa 10702 10721 SEQ ID NO: 2561ttgggaagaagaggcagct 12281 12300 1 3 SEQ ID NO: 2256 ttccaaaattgatgatatc10715 10734 SEQ ID NO: 2562 gatatacactagggaggaa 12737 12756 1 3 SEQ IDNO: 2257 gagaacatacaagcaaagc 10852 10871 SEQ ID NO: 2563gcttggttttgccagtctc 2459 2478 1 3 SEQ ID NO: 2258 atggcaaatgtcagctctt10889 10908 SEQ ID NO: 2564 aagaggtatttaaagccat 12952 12971 1 3 SEQ IDNO: 2259 tggcaaatgtcagctcttg 10890 10909 SEQ ID NO: 2565caagaggtatttaaagcca 12951 12970 1 3 SEQ ID NO: 2260 ttgttcaggtccatgcaag10906 10925 SEQ ID NO: 2566 cttgggggaggaggaacaa 14058 14077 1 3 SEQ IDNO: 2261 tgttcaggtccatgcaagt 10907 10926 SEQ ID NO: 2567acttgggggaggaggaaca 14057 14076 1 3 SEQ ID NO: 2262 agttccttccatgatttcc10932 10951 SEQ ID NO: 2568 ggaatctgatgaggaaact 12248 12267 1 3 SEQ IDNO: 2263 tgctaacactaagaaccag 10979 10998 SEQ ID NO: 2569ctggatgtaaccaccagca 11178 11197 1 3 SEQ ID NO: 2264 actaagaaccagaagatca10986 11005 SEQ ID NO: 2570 tgatcaagaacctgttagt 13339 13358 1 3 SEQ IDNO: 2265 ctaagaaccagaagatcag 10987 11006 SEQ ID NO: 2571ctgatcaagaacctgttag 13338 13357 1 3 SEQ ID NO: 2266 cagaagatcagatggaaaa10995 11014 SEQ ID NO: 2572 ttttcagaccaactctctg 13614 13633 1 3 SEQ IDNO: 2267 aaaaatgaagtccggattc 11010 11029 SEQ ID NO: 2573gaatttgaaagttcgtttt 9272 9291 1 3 SEQ ID NO: 2268 gattcattctgggtctttc11024 11043 SEQ ID NO: 2574 gaaaacctatgccttaatc 13158 13177 1 3 SEQ IDNO: 2269 aagaaaaggcacaccttga 11071 11090 SEQ ID NO: 2575tcaaaacctactgtctctt 10458 10477 1 3 SEQ ID NO: 2270 aaggacacctaaggttcct11107 11126 SEQ ID NO: 2576 aggacaccaaaataacctt 7564 7583 1 3 SEQ ID NO:2271 ccagcattggtaggagaca 11191 11210 SEQ ID NO: 2577 tgtcaacaagtaccactgg12362 12381 1 3 SEQ ID NO: 2272 ctttgtgtacaccaaaaac 11231 11250 SEQ IDNO: 2578 gtttttaaattgttgaaag 13140 13159 1 3 SEQ ID NO: 2273ccatccctgtaaaagtttt 11269 11288 SEQ ID NO: 2579 aaaagggtcatggaaatgg 88858904 1 3 SEQ ID NO: 2274 tgatctaaattcagttctt 11324 11343 SEQ ID NO: 2580aagatagtcagtctgatca 13326 13345 1 3 SEQ ID NO: 2275 aagaagctgagaacttcat11424 11443 SEQ ID NO: 2581 atgagatcaacacaatctt 13102 13121 1 3 SEQ IDNO: 2276 tttgccctcaacctaccaa 11445 11464 SEQ ID NO: 2582ttggtacgagttactcaaa 12633 12652 1 3 SEQ ID NO: 2277 cttgattcccttttttgag11528 11547 SEQ ID NO: 2583 ctcaattttgattttcaag 8520 8539 1 3 SEQ ID NO:2278 ttcacgcttccaaaaagtg 11583 11602 SEQ ID NO: 2584 cactcattgattttctgaa12685 12704 1 3 SEQ ID NO: 2279 tgtttcagatggcattgct 11600 11519 SEQ IDNO: 2585 agcagattatgttgaaaca 11825 11844 1 3 SEQ ID NO: 2280aatgcagtagccaacaaga 11631 11650 SEQ ID NO: 2586 tcttttcagcccagccatt 92239242 1 3 SEQ ID NO: 2281 ctgagcagaccattgagat 11683 11702 SEQ ID NO: 2587atctgatgaggaaactcag 12251 12270 1 3 SEQ ID NO: 2282 tgagcagaccattgagatt11684 11703 SEQ ID NO: 2588 aatctgatgaggaaactca 12250 12269 1 3 SEQ IDNO: 2283 ttgagattccctccattaa 11695 11714 SEQ ID NO: 2589ttaatcttcataagttcaa 13171 13190 1 3 SEQ ID NO: 2284 acttggagtgccagtttga11799 11818 SEQ ID NO: 2590 tcaattgggagagacaagt 6496 6515 1 3 SEQ ID NO:2285 caaatttgaaggacttcag 11996 12015 SEQ ID NO: 2591 ctgagaacttcatcatttg11430 11449 1 3 SEQ ID NO: 2286 agcccagcgttcaccgatc 12048 12067 SEQ IDNO: 2592 gatccaagtatagttggct 13278 13297 1 3 SEQ ID NO: 2287cagcgttcaccgatctcca 12052 12071 SEQ ID NO: 2593 tggacctgcaccaaagctg13952 13971 1 3 SEQ ID NO: 2288 ctccatctgcgctaccaga 12066 12085 SEQ IDNO: 2594 tctgatatacatcacggag 13703 13722 1 3 SEQ ID NO: 2289atgaggaaactcagatcaa 12256 12275 SEQ ID NO: 2595 ttgagttgcccaccatcat11659 11678 1 3 SEQ ID NO: 2290 aggcagcttctggcttgct 12292 12311 SEQ IDNO: 2596 agcaagtctttcctggcct 3010 3029 1 3 SEQ ID NO: 2291tgaaagacaacgtgcccaa 12319 12338 SEQ ID NO: 2597 ttgggagagacaagtttca 65006519 1 3 SEQ ID NO: 2292 tatgattatgtcaacaagt 12354 12373 SEQ ID NO: 2598actttgcactatgttcata 12755 12774 1 3 SEQ ID NO: 2293 cattaggcaaattgatgat12467 12486 SEQ ID NO: 2599 atcaacacaatcttcaatg 13107 13126 1 3 SEQ IDNO: 2294 ttgactcaggaaggccaag 12576 12595 SEQ ID NO: 2600cttggtacgagttactcaa 12632 12651 1 3 SEQ ID NO: 2295 gaaacctgggatatacact12728 12747 SEQ ID NO: 2601 agtgattacacttcctttc 12857 12876 1 3 SEQ IDNO: 2296 tcctttcgagttaaggaaa 12869 12888 SEQ ID NO: 2602tttctgccactgctcagga 13516 13535 1 3 SEQ ID NO: 2297 gccattcagtctctcaaga12966 12985 SEQ ID NO: 2603 tcttccgttctgtaatggc 5794 5813 1 3 SEQ ID NO:2298 gtgctacgtaatcttcagg 12993 13012 SEQ ID NO: 2604 cctgcaccaaagctggcac13956 13975 1 3 SEQ ID NO: 2299 agctgaaagagatgaaatt 13057 13076 SEQ IDNO: 2605 aatttattcaaaacgagct 13192 13211 1 3 SEQ ID NO: 2300aatttacttatcttattaa 13072 13091 SEQ ID NO: 2606 ttaaaagaaatcttcaatt13807 13826 1 3 SEQ ID NO: 2301 ttttaaattgttgaaagaa 13142 13161 SEQ IDNO: 2607 ttctctctatgggaaaaaa 9558 9577 1 3 SEQ ID NO: 2302taatcttcataagttcaat 13172 13191 SEQ ID NO: 2608 attgagattccctccatta11694 11713 1 3 SEQ ID NO: 2303 atattttgatccaagtata 13271 13290 SEQ IDNO: 2609 tataagcagaagcacatat 13929 13948 1 3 SEQ ID NO: 2304tgaaatattatgaacttga 13303 13322 SEQ ID NO: 2610 tcaaccttaatgattttca 82878306 1 3 SEQ ID NO: 2305 caatttctgcacagaaata 13434 13453 SEQ ID NO: 2611tattcttcttttccaattg 13826 13845 1 3 SEQ ID NO: 2306 agaagattgcagagctttc13501 13520 SEQ ID NO: 2612 gaaatcttcaatttattct 13813 13832 1 3 SEQ IDNO: 2307 gaagaaaataatttctgat 13562 13581 SEQ ID NO: 2613atcagttcagataaacttc 7991 8010 1 3 SEQ ID NO: 2308 ttgacctgtccattcaaaa13672 13691 SEQ ID NO: 2614 ttttgagaatgaatttcaa 10414 10433 1 3 SEQ IDNO: 2309 tcaaaactaccacacattt 13685 13704 SEQ ID NO: 2615aaattccttgacatgttga 7362 7381 1 3 SEQ ID NO: 2310 ttttttaaaagaaatcttc13803 13822 SEQ ID NO: 2616 gaagtgtcagtggcaaaaa 10374 10393 1 3 SEQ IDNO: 2311 aggatctgagttattttgc 14035 14054 SEQ ID NO: 2617gcaagggttcactgttcct 7856 7875 1 3 SEQ ID NO: 2312 tttgctaaacttgggggag14049 14068 SEQ ID NO: 2618 ctccccaggacctttcaaa 9834 9853 1 3 # = MatchNumber B = Middle Matching Bases

TABLE 9 Selected palindromic sequences from human ApoB Start End StartEnd Source Index Index Match Index Index # B SEQ ID NO: 2619ggccattccagaagggaag 517 536 SEQ ID NO: 3948 cttccgttctgtaatggcc 58035822 1 9 SEQ ID NO: 2620 tgccatctcgagagttcca 4107 4162 SEQ ID NO: 3949tggaactctctccatggca 10884 10903 1 8 SEQ ID NO: 2621 catgtcaaacactttgtta7064 7083 SEQ ID NO: 3950 taacaaattccttgacatg 7366 7385 1 8 SEQ ID NO:2622 tttgttataaatcttattg 7076 7095 SEQ ID NO: 3951 caataagatcaatagcaaa8998 9017 1 8 SEQ ID NO: 2623 tctggaaaagggtcatgga 8888 8907 SEQ ID NO:3959 tccatgtcccatttacaga 11364 11383 1 8 SEQ ID NO: 2624cagctcttgttcaggtcca 10908 10927 SEQ ID NO: 3960 tggacctgcaccaaagctg13980 13979 1 8 SEQ ID NO: 2625 ggaggttccccagctctgc 364 383 SEQ ID NO:3961 gcagccctgggaaaactcc 6455 6474 1 7 SEQ ID NO: 2626ctgttttgaagactctcca 1089 1108 SEQ ID NO: 3962 tggagggtagtcataacag 1033510354 1 7 SEQ ID NO: 2627 agtggctgaaacgtgtgca 1305 1324 SEQ ID NO: 3963tgcagagctttctgccact 13516 13535 1 7 SEQ ID NO: 2628 ccaaaatagaagggaatct2076 2095 SEQ ID NO: 3964 agattcctttgccttttgg 4008 4027 1 7 SEQ ID NO:2629 tgaagagaagattgaattt 3628 3647 SEQ ID NO: 3965 aaattctcttttcttttca9220 9239 1 7 SEQ ID NO: 2630 agtggtggcaacaccagca 4238 4257 SEQ ID NO:3966 tgctagtgaggccaacact 10857 10676 1 7 SEQ ID NO: 2631aaggctccacaagtcatca 5958 5977 SEQ ID NO: 3967 tgatgatatctggaacctt 1073210751 1 7 SEQ ID NO: 2632 gtcagccaggtttatagca 7733 7752 SEQ ID NO: 3968tgctaagaaccttactgac 7789 7808 1 7 SEQ ID NO: 2633 tgatatctggaaccttgaa10735 10754 SEQ ID NO: 3969 ttcactgttcctgaaatca 7871 7890 1 7 SEQ ID NO:2634 gtcaagttgagcaatttct 13431 13450 SEQ ID NO: 3970 agaaaaggcacaccttgac11080 11099 1 7 SEQ ID NO: 2635 atccagatggaaaagggaa 13488 13507 SEQ IDNO: 3971 ttccaatttccctgtggat 3688 3707 1 7 SEQ ID NO: 2636atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 3972 acttcagagaaatacaaat 1140911428 4 6 SEQ ID NO: 2637 ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 3973ccagacttccgtttaccag 8243 8262 2 6 SEQ ID NO: 2638 accaggaggttcttcttca1737 1756 SEQ ID NO: 3974 tgaagtgtagtctcctggt 5097 5116 2 6 SEQ ID NO:2639 aaagaagttctgaaagaat 1964 1983 SEQ ID NO: 3975 attccatcacaaatccttt9669 9688 2 6 SEQ ID NO: 2640 gctacagcttatggctcca 3578 3597 SEQ ID NO:3976 tggatctaaatgcagtagc 11631 11650 2 6 SEQ ID NO: 2641atcaatattgatcaatttg 6422 6441 SEQ ID NO: 3977 caaagaagtcaagattgat 45614580 2 6 SEQ ID NO: 2642 gaattatcttttaaaacat 7334 7353 SEQ ID NO: 3978atgtgttaacaaaatattc 11602 11521 2 6 SEQ ID NO: 2643 cgaggcccgcgctgctggc138 157 SEQ ID NO: 3979 gccagaagtgagatcctcg 3515 3534 1 6 SEQ ID NO:2644 acaactatgaggctgagag 279 298 SEQ ID NO: 3980 ctctgagcaacaaatttgt10317 10336 1 6 SEQ ID NO: 2645 gctgagagttccagtggag 290 309 SEQ ID NO:3981 ctccatggcaaatgtcagc 10893 10912 1 6 SEQ ID NO: 2646tgaagaaaaccaagaactc 456 475 SEQ ID NO: 3982 gagtcattgaggttcttca 49374956 1 6 SEQ ID NO: 2647 cctacttacatcctgaaca 566 585 SEQ ID NO: 3983tgttcataagggaggtagg 12774 12793 1 6 SEQ ID NO: 2648 ctacttacatcctgaacat567 586 SEQ ID NO: 3984 atgttcataagggaggtag 12773 12792 1 6 SEQ ID NO:2649 gagacagaagaagccaagc 623 642 SEQ ID NO: 3985 gcttggttttgccagtctc2467 2486 1 6 SEQ ID NO: 2650 cactcactttaccgtcaag 679 698 SEQ ID NO:3986 cttgaacacaaagtcagtg 6008 6027 1 6 SEQ ID NO: 2651ctgatcagcagcagccagt 830 649 SEQ ID NO: 3987 actgggaagtgcttatcag 52455264 1 6 SEQ ID NO: 2652 actggacgctaagaggaag 862 881 SEQ ID NO: 3988cttccccaaagagaccagt 2898 2917 1 6 SEQ ID NO: 2653 agaggaagcatgtggcaga873 892 SEQ ID NO: 3989 tctggcatttactttctct 5929 5948 1 6 SEQ ID NO:2654 tgaagactctccaggaact 1095 1114 SEQ ID NO: 3990 agttgaaggagactattca7224 7243 1 6 SEQ ID NO: 2655 ctctgagcaaaatatccag 1129 1148 SEQ ID NO:3991 ctggttactgagctgagag 1169 1188 1 6 SEQ ID NO: 2656atgaagcagtcacatctct 1197 1216 SEQ ID NO: 3992 agagctgccagtccttcat 1002410043 1 6 SEQ ID NO: 2657 ttgccacagctgattgagg 1217 1236 SEQ ID NO: 3993cctcctacagtggtggcaa 4230 4249 1 6 SEQ ID NO: 2658 agctgattgaggtgtccag1224 1243 SEQ ID NO: 3994 ctggattccacatgcagct 11855 11874 1 6 SEQ ID NO:2659 tgctccactcacatcctcc 1286 1305 SEQ ID NO: 3995 ggaggctttaagttcagca7609 7628 1 6 SEQ ID NO: 2660 tgaaacgtgtgcatgccaa 1311 1330 SEQ ID NO:3996 ttgggagagacaagtttca 6508 6527 1 6 SEQ ID NO: 2661gacattgctaattacctga 1511 1530 SEQ ID NO: 3997 tcagaagctaagcaatgtc 72407259 1 6 SEQ ID NO: 2662 ttcttcttcagactttcct 1746 1765 SEQ ID NO: 3998aggagagtccaaattagaa 8506 8525 1 6 SEQ ID NO: 2663 ccaatatcttgaactcaga1911 1930 SEQ ID NO: 3999 tctgaattcattcaattgg 6493 6512 1 6 SEQ ID NO:2664 aaagttagtgaaagaagtt 1954 1973 SEQ ID NO: 4000 aactaccctcactgccttt2140 2159 1 6 SEQ ID NO: 2665 aagttagtgaaagaagttc 1955 1974 SEQ ID NO:4001 gaacctctggcatttactt 5924 5943 1 6 SEQ ID NO: 2666aaagaagttctgaaagaat 1964 1983 SEQ ID NO: 4002 attctctggtaactacttt 54905509 1 6 SEQ ID NO: 2667 tttggctataccaaagatg 2330 2349 SEQ ID NO: 4003catcttaggcactgacaaa 5005 5024 1 6 SEQ ID NO: 2668 tgttgagaagctgattaaa2389 2408 SEQ ID NO: 4004 tttagccatcggctcaaca 5708 5727 1 6 SEQ ID NO:2669 caggaagggctcaaagaat 2569 2588 SEQ ID NO: 4005 attcctttaacaattcctg9500 9519 1 6 SEQ ID NO: 2670 aggaagggctcaaagaatg 2570 2589 SEQ ID NO:4006 cattcctttaacaattcct 9499 9518 1 6 SEQ ID NO: 2671gaagggctcaaagaatgac 2572 2591 SEQ ID NO: 4007 gtcagtcttcaggctcttc 79227941 1 6 SEQ ID NO: 2672 caaagaatgacttttttct 2580 2599 SEQ ID NO: 4008agaaggatggcattttttg 14008 14027 1 6 SEQ ID NO: 2673 catggagaatgcctttgaa2611 2630 SEQ ID NO: 4009 ttcagagccaaagtccatg 7127 7146 1 6 SEQ ID NO:2674 ggagccaaggctggagtaa 2687 2706 SEQ ID NO: 4010 ttactccaacgccagctcc3058 3077 1 6 SEQ ID NO: 2675 tcattccttccccaaagag 2892 2911 SEQ ID NO:4011 ctctctggggcatctatga 5147 5166 1 6 SEQ ID NO: 2676acctatgagctccagagag 3173 3192 SEQ ID NO: 4012 ctctcaagaccacagaggt 1298413003 1 6 SEQ ID NO: 2677 gggcaaaacgtcttacaga 3373 3392 SEQ ID NO: 4013tctgaaagacaacgtgccc 12325 12344 1 6 SEQ ID NO: 2678 accctggacattcagaaca3395 3414 SEQ ID NO: 4014 tgttgctaaggttcagggt 5683 5702 1 6 SEQ ID NO:2679 atgggcgacctaagttgtg 3437 3456 SEQ ID NO: 4015 cacaaattagtttcaccat8949 8968 1 6 SEQ ID NO: 2680 gatgaagagaagattgaat 3826 3645 SEQ ID NO:4016 attccagcttccccacatc 8338 8357 1 6 SEQ ID NO: 2681caatgtagataccaaaaaa 3664 3683 SEQ ID NO: 4017 ttttttggaaatgccattg 86518670 1 6 SEQ ID NO: 2682 gtagataccaaaaaaatga 3668 3687 SEQ ID NO: 4018tcatgtgatgggtctctac 4379 4398 1 6 SEQ ID NO: 2683 gcttcagttcatttggact4517 4536 SEQ ID NO: 4019 agtcaagaaggacttaagc 5312 5331 1 6 SEQ ID NO:2684 tttgtttgtcaaagaagtc 4552 4571 SEQ ID NO: 4020 gacttcagagaaatacaaa11408 11427 1 6 SEQ ID NO: 2685 ttgtttgtcaaagaagtca 4553 4572 SEQ ID NO:4021 tgacttcagagaaatacaa 11407 11428 1 6 SEQ ID NO: 2686tggcaatgggaaactcgct 5854 5873 SEQ ID NO: 4022 agcgagaatcaccctgcca 82278246 1 6 SEQ ID NO: 2687 aacctctggcatttacttt 5925 5944 SEQ ID NO: 4023aaaggagatgtcaagggtt 10607 10626 1 6 SEQ ID NO: 2688 catttactttctctcatga5934 5953 SEQ ID NO: 4024 tcatttgaaagaataaatg 7034 7053 1 6 SEQ ID NO:2689 aaagtcagtgccctgctta 6017 6036 SEQ ID NO: 4025 taagaaccttactgacttt7792 7811 1 6 SEQ ID NO: 2690 tcccattttttgagacctt 6330 6349 SEQ ID NO:4026 aaggacttcaggaatggga 12012 12031 1 6 SEQ ID NO: 2691catcaatattgatcaattt 6421 6440 SEQ ID NO: 4027 aaattaaaaagtcttgatg 67406759 1 6 SEQ ID NO: 2692 taaagatagttatgattta 6673 6692 SEQ ID NO: 4028taaaccaaaacttggttta 9027 9046 1 6 SEQ ID NO: 2693 tattgatgaaatcattgaa6721 6740 SEQ ID NO: 4029 ttcaaagacttaaaaaata 8015 8034 1 6 SEQ ID NO:2694 atgatctacatttgtttat 6798 6817 SEQ ID NO: 4030 ataaagaaattaaagtcat7388 7407 1 6 SEQ ID NO: 2695 agagacacatacagaatat 6927 6946 SEQ ID NO:4031 atatattgtcagtgcctct 13390 13409 1 6 SEQ ID NO: 2696gacacatacagaatataga 6930 6949 SEQ ID NO: 4032 tctaaattcagttcttgtc 1133511354 1 6 SEQ ID NO: 2697 agcatgtcaaacactttgt 7062 7081 SEQ ID NO: 4033acaaagtcagtgccctgct 6015 6034 1 6 SEQ ID NO: 2698 tttttagaggaaaccaagg7523 7542 SEQ ID NO: 4034 cctttgtgtacaccaaaaa 11238 11257 1 6 SEQ ID NO:2599 ttttagaggaaaccaaggc 7524 7543 SEQ ID NO: 4035 gcctttgtgtacaccaaaa11237 11256 1 6 SEQ ID NO: 2700 ggaagatagacttcctgaa 9315 9334 SEQ ID NO:4036 ttcagaaatactgttttcc 12832 12851 1 6 SEQ ID NO: 2701cactgtttctgagtcccag 9342 9361 SEQ ID NO: 4037 ctgggacctaccaagagtg 1253112550 1 6 SEQ ID NO: 2702 cacaaatcctttggctgtg 9676 9695 SEQ ID NO: 4038cacatttcaaggaattgtg 10071 10090 1 6 SEQ ID NO: 2703 ttcctggatacactgttcc9861 9880 SEQ ID NO: 4039 ggaactgttgactcaggaa 12577 12596 1 6 SEQ ID NO:2704 gaaatctcaagctttctct 10050 10069 SEQ ID NO: 4040 agagccaggtcgagctttc11052 11071 1 6 SEQ ID NO: 2705 tttcttcatcttcatctgt 10218 10237 SEQ IDNO: 4041 acagctgaaagagatgaaa 13063 13082 1 6 SEQ ID NO: 2706tctaccgctaaaggagcag 10529 10548 SEQ ID NO: 4042 ctgcacgctttgaggtaga11769 11788 1 6 SEQ ID NO: 2707 ctaccgctaaaggagcagt 10530 10549 SEQ IDNO: 4043 actgcacgctttgaggtag 11768 11787 1 6 SEQ ID NO: 2708agggcctctttttcaccaa 10839 10858 SEQ ID NO: 4044 ttggccaggaagtggccct10965 10984 1 6 SEQ ID NO: 2709 ttctccatccctgtaaaag 11273 11292 SEQ IDNO: 4045 cttttcaccaacggagaa 10846 10865 1 6 SEQ ID NO: 2710gaaaaacaaagcagattat 11824 11843 SEQ ID NO: 4046 ataaactgcaagatttttc13608 13627 1 6 SEQ ID NO: 2711 actcactcattgattttct 12690 12709 SEQ IDNO: 4047 agaaaatcaggatctgagt 14035 14054 1 6 SEQ ID NO: 2712taaactaatagatgtaatc 12898 12917 SEQ ID NO: 4048 gattaccaccagcagttta13586 13605 1 6 SEQ ID NO: 2713 caaaacgagcttcaggaag 13208 13227 SEQ IDNO: 4049 cttcgtgaagaatattttg 13268 13287 1 6 SEQ ID NO: 2714tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4050 aacacttacttgaattcca 1067010689 3 5 SEQ ID NO: 2715 gatttgaaatccaaagaag 2408 2427 SEQ ID NO: 4051cttcagagaaatacaaatc 11410 11429 3 5 SEQ ID NO: 2716 atttgaaatccaaagaagt2409 2428 SEQ ID NO: 4052 acttcagagaaatacaaat 11409 11428 3 5 SEQ ID NO:2717 atcaacagccgcttctttg 998 1017 SEQ ID NO: 4053 caaagaagtcaagattgat4561 4580 2 5 SEQ ID NO: 2718 tgttttgaagactctccag 1090 1109 SEQ ID NO:4054 ctggaaagttaaaacaaca 6963 6982 2 5 SEQ ID NO: 2719cccttctgatagatgtggt 1332 1351 SEQ ID NO: 4055 accaaagctggcaccaggg 1396913988 2 5 SEQ ID NO: 2720 tgagcaagtgaagaacttt 1876 1895 SEQ ID NO: 4056aaagccattcagtctctca 12971 12990 2 5 SEQ ID NO: 2721 atttgaaatccaaagaagt2409 2428 SEQ ID NO: 4057 acttttctaaacttgaaat 9063 9082 2 5 SEQ ID NO:2722 atccaaagaagtcccggaa 2416 2435 SEQ ID NO: 4058 ttccggggaaacctgggat12729 12748 2 5 SEQ ID NO: 2723 agagcctacctccgcatct 2438 2457 SEQ ID NO:4059 agatggtacgttagcctct 11929 11948 2 5 SEQ ID NO: 2724aatgcctttgaactcccca 2618 2837 SEQ ID NO: 4060 tgggaactacaatttcatt 70207039 2 5 SEQ ID NO: 2725 gaagtccaaattccggatt 3305 3324 SEQ ID NO: 4061aatcttcaatttattcttc 13823 13842 2 5 SEQ ID NO: 2726 tgcaagcagaagccagaag3504 3523 SEQ ID NO: 4062 cttcaggttccatcgtgca 11384 11403 2 5 SEQ ID NO:2727 gaagagaagattgaatttg 3629 3648 SEQ ID NO: 4063 caaaacctactgtctcttc10467 10486 2 5 SEQ ID NO: 2728 atgctaaaggcacatatgg 4605 4624 SEQ ID NO:4064 ccatatgaaagtcaagcat 12664 12683 2 5 SEQ ID NO: 2729tccctcacctccacctctg 4745 4764 SEQ ID NO: 4065 cagattctcagatgaggga 89208939 2 5 SEQ ID NO: 2730 atttacagctctgacaagt 5435 5454 SEQ ID NO: 4066acttttctaaacttgaaat 9063 9082 2 5 SEQ ID NO: 2731 aggagcctaccaaaataat5602 5521 SEQ ID NO: 4067 attatgttgaaacagtcct 11838 11857 2 5 SEQ ID NO:2732 aaagctgaagcacatcaat 6409 6428 SEQ ID NO: 4068 attgttgctcatctccttt10202 10221 2 5 SEQ ID NO: 2733 ctgctggaaacaacgagaa 9426 9445 SEQ ID NO:4069 ttctgattaccaccagcag 13582 13601 2 5 SEQ ID NO: 2734ttgaaggaattcttgaaaa 9590 9609 SEQ ID NO: 4070 ttttaaaagaaatcttcaa 1381313832 2 5 SEQ ID NO: 2735 gaagtaaaagaaaattttg 10751 10770 SEQ ID NO:4071 caaaacctactgtctcttc 10467 10486 2 5 SEQ ID NO: 2736tgaagaagatggcaaattt 11992 12011 SEQ ID NO: 4072 aaatgtcagctcttgttca10902 10921 2 5 SEQ ID NO: 2737 aggatctgagttattttgc 14043 14062 SEQ IDNO: 4073 gcaagtcagcccagttcct 10928 10947 2 5 SEQ ID NO: 2738gtgcccttctcggttgctg 26 45 SEQ ID NO: 4074 cagccattgacatgagcac 5748 57671 5 SEQ ID NO: 2739 ggcgctgcctgcgctgctg 154 173 SEQ ID NO: 4075cagctccacagactccgcc 3070 3089 1 5 SEQ ID NO: 2740 ctgcgctgctgctgctgct162 181 SEQ ID NO: 4076 agcagaaggtgcgaagcag 3232 3251 1 5 SEQ ID NO:2741 gctgctggcgggcgccagg 178 197 SEQ ID NO: 4077 cctggattccacatgcagc11854 11873 1 5 SEQ ID NO: 2742 aagaggaaatgctggaaaa 201 220 SEQ ID NO:4078 tttttcttcactacatctt 2592 2611 1 5 SEQ ID NO: 2743ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 4079 ccagacttccacatcccag 39233942 1 5 SEQ ID NO: 2744 tggagtccctgggactgct 304 323 SEQ ID NO: 4080agcatgcctagtttctcca 9953 9972 1 5 SEQ ID NO: 2745 ggagtccctgggactgctg305 324 SEQ ID NO: 4081 cagcatgcctagtttctcc 9952 9971 1 5 SEQ ID NO:2746 tgggactgctgattcaaga 313 332 SEQ ID NO: 4082 tcttccatcacttgaccca2050 2069 1 5 SEQ ID NO: 2747 ctgctgattcaagaagtgc 318 337 SEQ ID NO:4083 gcacaccttgacattgcag 11087 11106 1 5 SEQ ID NO: 2748tgccaccaggatcaactgc 334 353 SEQ ID NO: 4084 gcaggctgaactggtggca 27252744 1 5 SEQ ID NO: 2749 gccaccaggatcaactgca 335 354 SEQ ID NO: 4085tgcaggctgaactggtggc 2724 2743 1 5 SEQ ID NO: 2750 tgcaaggttgagctggagg350 369 SEQ ID NO: 4086 cctccacctctgatctgca 4752 4771 1 5 SEQ ID NO:2751 caaggttgagctggaggtt 352 371 SEQ ID NO: 4089 aacccctacatgaagcttg13763 13782 1 5 SEQ ID NO: 2752 ctctgcagcttcatcctga 377 396 SEQ ID NO:4090 tcaggaagcttctcaagag 13219 13238 1 5 SEQ ID NO: 2753cagcttcatcctgaagccc 382 401 SEQ ID NO: 4091 ggtcttgagttaaatgctg 49855004 1 5 SEQ ID NO: 2754 gcttcatcctgaagaccag 384 403 SEQ ID NO: 4092ctggacgctaagaggaagc 863 882 1 5 SEQ ID NO: 2755 tcatcctgaagaccagcca 387406 SEQ ID NO: 4093 tggcatggcattatgatga 3612 3631 1 5 SEQ ID NO: 2756gaaaaccaagaactctgag 460 479 SEQ ID NO: 4094 ctcaaccttaatgattttc 82948313 1 5 SEQ ID NO: 2757 agaactctgaggagtttgc 468 487 SEQ ID NO: 4095gcaagctatacagtattct 8385 8404 1 5 SEQ ID NO: 2758 tctgaggagtttgctgcag473 492 SEQ ID NO: 4096 ctgcaggggatcccccaga 2534 2553 1 5 SEQ ID NO:2759 tttgctgcagccatgtcca 482 501 SEQ ID NO: 4097 tggaagtgtcagtggcaaa10380 10399 1 5 SEQ ID NO: 2760 caagaggggcatcatttct 586 605 SEQ ID NO:4098 agaataaatgacgttcttg 7043 7062 1 5 SEQ ID NO: 2761tcactttaccgtcaagacg 682 701 SEQ ID NO: 4099 cgtctacactatcatgtga 43684387 1 5 SEQ ID NO: 2762 tttaccgtcaagacgagga 686 705 SEQ ID NO: 4100tccttgacatgttgataaa 7374 7393 1 5 SEQ ID NO: 2763 cactggacgctaagaggaa861 880 SEQ ID NO: 4101 ttccagaaagcagccagtg 12506 12525 1 5 SEQ ID NO:2764 aggaagcatgtggcagaag 875 894 SEQ ID NO: 4102 cttcatacacattaatcct9996 10015 1 5 SEQ ID NO: 2765 caaggagcaacacctcttc 901 920 SEQ ID NO:4103 gaagtagtactgcatcttg 6843 6862 1 5 SEQ ID NO: 2766acagactttgaaacttgaa 967 986 SEQ ID NO: 4104 ttcaattcttcaatgctgt 1050810527 1 5 SEQ ID NO: 2767 tgatgaagcagtcacatct 1195 1214 SEQ ID NO: 4105agatttgaggattccatca 7984 8003 1 5 SEQ ID NO: 2768 agcagtcacatctctcttg1201 1220 SEQ ID NO: 4106 caaggagaaactgactgct 6532 6551 1 5 SEQ ID NO:2769 ccagccccatcactttaca 1239 1258 SEQ ID NO: 4107 tgtagtctcctggtgctgg5102 5121 1 5 SEQ ID NO: 2770 ctccactcacatcctccag 1288 1307 SEQ ID NO:4108 ctggagcttagtaatggag 8717 8736 1 5 SEQ ID NO: 2771catgccaacccccttctga 1322 1341 SEQ ID NO: 4109 tcagatgagggaacacatg 89278946 1 5 SEQ ID NO: 2772 gagagatcttcaacatggc 1398 1417 SEQ ID NO: 4110gccaccctggaactctctc 10877 10898 1 5 SEQ ID NO: 2773 tcaacatggcgagggatca1407 1426 SEQ ID NO: 4111 tgatcccacctctcattga 2973 2992 1 5 SEQ ID NO:2774 ccaccttgtatgcgctgag 1437 1456 SEQ ID NO: 4112 ctcagggatctgaaggtgg8195 8214 1 5 SEQ ID NO: 2775 gtcaacaactatcataaga 1463 1482 SEQ ID NO:4113 tcttgagttaaatgctgac 4987 5006 1 5 SEQ ID NO: 2776tggacattgctaattacct 1509 1528 SEQ ID NO: 4114 aggtatattcgaaagtcca 1280712828 1 5 SEQ ID NO: 2777 ggacattgctaattacctg 1510 1529 SEQ ID NO: 4115caggtatattcgaaagtcc 12806 12825 1 5 SEQ ID NO: 2778 ttctgcgggtcattggaaa1581 1600 SEQ ID NO: 4116 tttcacatgccaaggagaa 6522 6641 1 5 SEQ ID NO:2779 ccagaactcaagtcttcaa 1628 1647 SEQ ID NO: 4117 ttgaagtgtagtctcctgg5096 5115 1 5 SEQ ID NO: 2780 agtcttcaatcctgaaatg 1638 1557 SEQ ID NO:4118 catttctgattggtggact 7765 7784 1 5 SEQ ID NO: 2781tgagcaagtgaagaacttt 1876 1895 SEQ ID NO: 4119 aaagtgccacttttactca 61916210 1 5 SEQ ID NO: 2782 agcaagtgaagaactttgt 1878 1897 SEQ ID NO: 4120acaaagtcagtgccctgct 6015 6034 1 5 SEQ ID NO: 2783 tctgaaagaatctcaactt1972 1991 SEQ ID NO: 4121 aagtccataatggttcaga 12819 12838 1 5 SEQ ID NO:2784 actgtcatggacttcagaa 1994 2013 SEQ ID NO: 4122 ttctgaatatatttgtcagt13384 13403 1 5 SEQ ID NO: 2785 acttgacccagcctcagcc 2069 2078 SEQ ID NO:4123 ggctcaccctgagagaagt 12399 12418 1 5 SEQ ID NO: 2786tccaaataactaccttcct 2104 2123 SEQ ID NO: 4124 aggaagatatgaagatgga 47204739 1 5 SEQ ID NO: 2787 actaccctcactgcctttg 2141 2160 SEQ ID NO: 4125caaatttgtggagggtagt 10327 10346 1 5 SEQ ID NO: 2788 ttggatttgcttcagctga2157 2176 SEQ ID NO: 4126 tcagtataagtacaaccaa 9400 9419 1 5 SEQ ID NO:2789 ttggaagctctttttggga 2219 2238 SEQ ID NO: 4127 tcccgattcacgcttccaa11585 11604 1 5 SEQ ID NO: 2790 ggaagctctttttgggaag 2221 2240 SEQ ID NO:4128 cttcagaaagctaccttcc 7937 7955 1 5 SEQ ID NO: 2791tttttcccagacagtgtca 2246 2265 SEQ ID NO: 4129 tgaccttctctaagcaaaa 48844903 1 5 SEQ ID NO: 2792 agacagtgtcaacaaagct 2254 2273 SEQ ID NO: 4130agcttggttttgccagtct 2466 2485 1 5 SEQ ID NO: 2793 ctttggctataccaaagat2329 2348 SEQ ID NO: 4131 atctcgtgtctaggaaaag 5976 5995 1 5 SEQ ID NO:2794 caaagatgataaacatgag 2341 2360 SEQ ID NO: 4132 ctcaaggataacgtgtttg12617 12636 1 5 SEQ ID NO: 2795 gatatggtaaatggaataa 2363 2382 SEQ ID NO:4133 ttatcttattaattatatc 13087 13106 1 5 SEQ ID NO: 2796ggaataatgctcagtgttg 2375 2394 SEQ ID NO: 4134 caacacttacttgaattcc 1066910688 1 5 SEQ ID NO: 2797 tttgaaatccaaagaagtc 2410 2429 SEQ ID NO: 4135gacttcagagaaatacaaa 11408 11427 1 5 SEQ ID NO: 2798 gatcccccagatgattgga2542 2561 SEQ ID NO: 4136 tccaatttccctgtggatc 3669 3708 1 5 SEQ ID NO:2799 cagatgattggagaggtca 2549 2568 SEQ ID NO: 4137 tgaccacacaaacagtctg5371 5390 1 5 SEQ ID NO: 2800 agaatgacttttttcttca 2583 2602 SEQ ID NO:4138 tgaagtccggattcattct 11023 11042 1 5 SEQ ID NO: 2801gaactccccactggagctg 2627 2646 SEQ ID NO: 4139 cagctcaaccgtacagttc 1186911688 1 5 SEQ ID NO: 2802 atatcttcatctggagtca 2660 2679 SEQ ID NO: 4140tgacttcagtgcagaatat 11974 11993 1 5 SEQ ID NO: 2803 gtcattgctcccggagcca2675 2694 SEQ ID NO: 4141 tggccccgtttaccatgac 5817 5836 1 5 SEQ ID NO:2804 gctgaagtttatcattcct 2881 2900 SEQ ID NO: 4142 aggaggctttaagttcagc7608 7627 1 5 SEQ ID NO: 2805 attccttccccaaagagac 2894 2913 SEQ ID NO:4143 gtctcttcctccatggaat 10478 10497 1 5 SEQ ID NO: 2806ctcattgagaacaggcagt 2984 3003 SEQ ID NO: 4144 actgactgcacgctttgag 1176411783 1 5 SEQ ID NO: 2807 ttgagcagtattctgtcag 3150 3169 SEQ ID NO: 4145ctgagagaagtgtcttcaa 12407 12426 1 5 SEQ ID NO: 2808 accttgtccagtgaagtcc3293 3312 SEQ ID NO: 4146 ggacggtactgtcccaggt 12792 12811 1 5 SEQ ID NO:2809 ccagtgaagtccaaattcc 3300 3319 SEQ ID NO: 4147 ggaaggcagagtttactgg9156 9175 1 5 SEQ ID NO: 2810 acattcagaacaagaaaat 3402 3421 SEQ ID NO:4148 atttcctaaagctggatgt 11175 11194 1 5 SEQ ID NO: 2811gaaaaatcaagggtgttat 3471 3490 SEQ ID NO: 4149 ataaactgcaagatttttc 1360813627 1 5 SEQ ID NO: 2812 aaatcaagggtgttatttc 3474 3493 SEQ ID NO: 4150gaaacaatgcattagattt 9753 9772 1 5 SEQ ID NO: 2813 tggcattatgatgaagaga3617 3636 SEQ ID NO: 4151 tctcccgtgtataatgcca 11789 11808 1 5 SEQ ID NO:2814 aagagaagattgaatttga 3630 3649 SEQ ID NO: 4152 tcaaaacctactgtctctt10466 10485 1 5 SEQ ID NO: 2815 aaatgacttccaatttccc 3681 3700 SEQ ID NO:4153 gggaactacaatttcattt 7021 7040 1 5 SEQ ID NO: 2816atgacttccaatttccctg 3683 3702 SEQ ID NO: 4154 caggctgattacgagtcat 49254944 1 5 SEQ ID NO: 2817 acttccaatttccctgtgg 3686 3705 SEQ ID NO: 4155ccacgaaaaatatggaagt 10368 10387 1 5 SEQ ID NO: 2818 agttgcaatgagctcatgg3811 3830 SEQ ID NO: 4156 ccatcagttcagataaact 7997 8016 1 5 SEQ ID NO:2819 tttgcaagaccacctcaat 3868 3887 SEQ ID NO: 4157 attgacctgtccattcaaa13679 13698 1 5 SEQ ID NO: 2820 gaaggagttcaacctccag 3892 3911 SEQ ID NO:4158 ctggaattgtcattccttc 11738 11755 1 5 SEQ ID NO: 2821acttccacatcccagaaaa 3927 3946 SEQ ID NO: 4159 ttttaacaaaagtggaagt 68296848 1 5 SEQ ID NO: 2822 ctcttcttaaaaagcgatg 3947 3966 SEQ ID NO: 4160catcactgccaaaggagag 8494 8513 1 5 SEQ ID NO: 2823 aaaagcgatggccgggtca3956 3975 SEQ ID NO: 4161 tgactcactcattgatttt 12688 12707 1 5 SEQ ID NO:2824 ttcctttgccttttggtgg 4011 4030 SEQ ID NO: 4162 ccacaaacaatgaagggaa9264 9283 1 5 SEQ ID NO: 2825 caagtctgtgggattccat 4087 4106 SEQ ID NO:4163 atgggaaaaaacaggcttg 9574 9593 1 5 SEQ ID NO: 2826aagtccctacttttaccat 4125 4144 SEQ ID NO: 4164 atgggaagtataagaactt 48424861 1 5 SEQ ID NO: 2827 tgcctctcctgggtgttct 4167 4186 SEQ ID NO: 4165agaaaaacaaacacaggca 9651 9670 1 5 SEQ ID NO: 2828 accagcacagaccatttca4250 4269 SEQ ID NO: 4166 tgaagtgtagtctcctggt 5097 5116 1 5 SEQ ID NO:2829 ccagcacagaccatttcag 4251 4270 SEQ ID NO: 4167 ctgaaatacaatgctctgg5519 5538 1 5 SEQ ID NO: 2830 actatcatgtgatgggtct 4375 4394 SEQ ID NO:4168 agacacctgattttatagt 7956 7975 1 5 SEQ ID NO: 2831accacagatgtctgcttca 4504 4523 SEQ ID NO: 4169 tgaaggctgactctgtggt 42904309 1 5 SEQ ID NO: 2832 ccacagatgtctgcttcag 4505 4524 SEQ ID NO: 4170ctgagcaacaaatttgtgg 10319 10338 1 5 SEQ ID NO: 2833 tttggactccaaaaagaaa4528 4547 SEQ ID NO: 4171 tttctctcatgattacaaa 5941 5960 1 5 SEQ ID NO:2834 tcaaagaagtcaagattga 4560 4579 SEQ ID NO: 4172 tcaaggataacgtgtttga12618 12637 1 5 SEQ ID NO: 2835 atgagaactacgagctgac 4806 4825 SEQ ID NO:4173 gtcagatattgttgctcat 10195 10214 1 5 SEQ ID NO: 2836ttaaaatctgacaccaatg 4826 4845 SEQ ID NO: 4174 cattcattgaagatgttaa 73507369 1 5 SEQ ID NO: 2837 gaagtataagaactttgcc 4840 4865 SEQ ID NO: 4175ggcaaatttgaaggacttc 12002 12021 1 5 SEQ ID NO: 2838 aagtataagaactttgcca4847 4866 SEQ ID NO: 4176 tggcaaatttgaaggactt 12001 12020 1 5 SEQ ID NO:2839 ttcttcagcctgctttctg 4949 4968 SEQ ID NO: 4177 cagaatccagatacaagaa6892 6911 1 5 SEQ ID NO: 2840 ctggatcactaaattccca 4965 4984 SEQ ID NO:4178 tgggtctttccagagccag 11041 11060 1 5 SEQ ID NO: 2841aaattaatagtggtgctca 5022 5041 SEQ ID NO: 4179 tgagaagccccaagaattt 62566275 1 5 SEQ ID NO: 2842 agtgcaacgaccaacttga 5081 5100 SEQ ID NO: 4180tcaaattcctggatacact 9856 9875 1 5 SEQ ID NO: 2843 ctgggaagtgcttatcagg5246 5265 SEQ ID NO: 4181 cctgaccttcacataccag 8318 8337 1 5 SEQ ID NO:2844 gcaaaaacattttcaactt 5286 5305 SEQ ID NO: 4182 aagtaaaagaaaattttgc10752 10771 1 5 SEQ ID NO: 2845 aaaaacattttcaacttca 5288 5307 SEQ ID NO:4183 tgaagtaaaagaaaatttt 10750 10769 1 5 SEQ ID NO: 2846tcagtcaagaaggacttaa 5310 5329 SEQ ID NO: 4184 ttaaggacttccattctga 1337113390 1 5 SEQ ID NO: 2847 tcaaatgacatgatgggct 5333 5352 SEQ ID NO: 4185agcccatcaatatcattga 6213 6232 1 5 SEQ ID NO: 2848 cacacaaacagtctgaaca5375 5394 SEQ ID NO: 4186 tgtttcaactgcctttgtg 11227 11246 1 5 SEQ ID NO:2849 tcttcaaaacttgacaaca 5417 5436 SEQ ID NO: 4187 tgttttcctatttccaaga12843 12862 1 5 SEQ ID NO: 2850 caagttttataagcaaact 5449 5468 SEQ ID NO:4188 agttattttgctaaacttg 14061 14070 1 5 SEQ ID NO: 2851tggtaactactttaaacag 5496 5515 SEQ ID NO: 4189 ctgtttttagaggaaacca 75207539 1 5 SEQ ID NO: 2852 aacagtgacctgaaataca 5510 5529 SEQ ID NO: 4190tgtatagcaaattcctgtt 5898 5917 1 5 SEQ ID NO: 2853 gggaaactacggctagaac5552 5571 SEQ ID NO: 4191 gttccttccatgatttccc 10941 10960 1 5 SEQ ID NO:2854 aacacatctatgccatctc 5628 5647 SEQ ID NO: 4192 gagacagcatcttcgtgtt11212 11231 1 5 SEQ ID NO: 2855 tcagcaagctataaagcag 5660 5679 SEQ ID NO:4193 ctgctaagaaccttactga 7788 7807 1 5 SEQ ID NO: 2856gcagacactgttgctaagg 5675 5694 SEQ ID NO: 4194 cctttcaagcactgactgc 1175411773 1 5 SEQ ID NO: 2857 tctggggagaacatactgg 5874 5893 SEQ ID NO: 4195ccaggttttccacaccaga 8046 8065 1 5 SEQ ID NO: 2858 ttctctcatgattacaaag5942 5961 SEQ ID NO: 4196 ctttttcaccaacggagaa 10846 10865 1 5 SEQ ID NO:2859 ctgagcagacaggcacctg 6042 6061 SEQ ID NO: 4197 caggaggctttaagttcag7607 7626 1 5 SEQ ID NO: 2860 caatttaacaacaatgaat 6074 6093 SEQ ID NO:4198 attccttcctttacaattg 8090 8109 1 5 SEQ ID NO: 2861tggacgaactctggctgac 6148 6167 SEQ ID NO: 4199 gtcagcccagttccttcca 1093210951 1 5 SEQ ID NO: 2862 cttttactcagtgagccca 6200 6219 SEQ ID NO: 4200tgggctaaacgtatgaaag 7835 7854 1 5 SEQ ID NO: 2863 tcattgatgctttagagat6225 6244 SEQ ID NO: 4201 atcttcataagttcaatga 13182 13201 1 5 SEQ ID NO:2864 aaaaccaagatgttcactc 6303 6322 SEQ ID NO: 4202 gagtgaaatgctgtttttt8638 8657 1 5 SEQ ID NO: 2865 aggaatcgacaaaccatta 6365 6384 SEQ ID NO:4203 taatgattttcaagttcct 8302 8321 1 5 SEQ ID NO: 2866tagttgtactggaaaacgt 6384 6403 SEQ ID NO: 4204 acgttagcctctaagacta 1193611955 1 5 SEQ ID NO: 2867 ggaaaacgtacagagaaag 6394 6413 SEQ ID NO: 4205cttttacaattcattttcc 13022 13041 1 5 SEQ ID NO: 2868 gaaaacgtacagagaaagc6395 6414 SEQ ID NO: 4206 gctttctcttccacatttc 10060 10079 1 5 SEQ ID NO:2869 aaagctgaagcacatcaat 6409 6428 SEQ ID NO: 4207 attgatgttagagtgcttt6992 7011 1 5 SEQ ID NO: 2870 aagctgaagcacatcaata 6410 6429 SEQ ID NO:4208 tattgatgttagagtgctt 6991 7010 1 5 SEQ ID NO: 2871tgaagcacatcaatattga 6414 6433 SEQ ID NO: 4209 tcaaccttaatgattttca 62958314 1 5 SEQ ID NO: 2872 atcaatattgatcaatttg 6422 6441 SEQ ID NO: 4210caaagccatcactgatgat 1668 1687 1 5 SEQ ID NO: 2873 taatgattatctgaattca6464 6503 SEQ ID NO: 4211 tgaaatcattgaaaaatta 6727 6746 1 5 SEQ ID NO:2874 gattatctgaattcattca 6466 6507 SEQ ID NO: 4212 tgaagtagctgagaaaatc7102 7121 1 5 SEQ ID NO: 2875 aattgggagagacaagttt 6508 6525 SEQ ID NO:4213 aaacattcctttaacaatt 9498 9515 1 5 SEQ ID NO: 2876aaaatagctattgctaata 6701 6720 SEQ ID NO: 4214 tattgaaaatattgatttt 68146833 1 5 SEQ ID NO: 2877 aaaattaaaaagtcttgat 6739 6758 SEQ ID NO: 4215atcatatccgtgtaatttt 6765 6784 1 5 SEQ ID NO: 2878 ttgaaaatattgattttaa6818 6835 SEQ ID NO: 4216 ttaatcttcataagttcaa 13179 13198 1 5 SEQ ID NO:2879 agacatccagcacctagct 6946 6965 SEQ ID NO: 4217 agcttggttttgccagtct2468 2485 1 5 SEQ ID NO: 2880 caatttcatttgaaagaat 7029 7048 SEQ ID NO:4218 attccttcctttacaattg 8090 8109 1 5 SEQ ID NO: 2881aggttttaatggataaatt 7182 7201 SEQ ID NO: 4219 aattgttgaaagaaaacct 1315513174 1 5 SEQ ID NO: 2882 cagaagctaagcaatgtcc 7241 7280 SEQ ID NO: 4220ggacaaggcccagaatctg 12553 12572 1 5 SEQ ID NO: 2883 taagataaaagattacttt7270 7289 SEQ ID NO: 4221 aaagaaaacctatgcctta 13163 13182 1 5 SEQ ID NO:2884 aaagattactttgagaaat 7277 7296 SEQ ID NO: 4222 atttcttaaacattccttt9489 9508 1 5 SEQ ID NO: 2885 gagaaattagttggattta 7289 7308 SEQ ID NO:4223 taaagccattcagtctctc 12970 12989 1 5 SEQ ID NO: 2886atttattgatgatgctgtc 7303 7322 SEQ ID NO: 4224 gacatgttgataaagaaat 73797398 1 5 SEQ ID NO: 2887 gaattatcttttaaaacat 7334 7353 SEQ ID NO: 4225atgtatcaaatggacattc 7685 7704 1 5 SEQ ID NO: 2888 ttaccaccagtttgtagat7411 7430 SEQ ID NO: 4226 atctggaaccttgaagtaa 10739 10758 1 5 SEQ ID NO:2889 ttgcagtgtatctggaaag 7548 7567 SEQ ID NO: 4227 cttttcacattagatgcaa8420 8439 1 5 SEQ ID NO: 2890 cattcagcaggaacttcaa 7899 7718 SEQ ID NO:4228 ttgaaggacttcaggaatg 12009 12028 1 5 SEQ ID NO: 2891acacctgattttatagtcc 7958 7977 SEQ ID NO: 4229 ggactcaaggataacgtgt 1281412633 1 5 SEQ ID NO: 2892 ggattccatcagttcagat 7992 8011 SEQ ID NO: 4230atcttcaatgattatatcc 13124 13143 1 5 SEQ ID NO: 2893 ttgtagaaatgaaagtaaa8112 8131 SEQ ID NO: 4231 tttatgattatgtcaacaa 12380 12379 1 5 SEQ ID NO:2894 ctgaacagtgagctgcagt 8168 8175 SEQ ID NO: 4232 actggacttctctagtcag8800 8828 1 5 SEQ ID NO: 2895 aatccaatctcctcttttc 8407 8426 SEQ ID NO:4233 gaaaaatgaagtccggatt 11017 11036 1 5 SEQ ID NO: 2896attttgattttcaagcaaa 8532 8551 SEQ ID NO: 4234 tttgcaagttaaagaaaat 1402314042 1 5 SEQ ID NO: 2897 ttttgattttcaagcaaat 8533 8552 SEQ ID NO: 4235atttgatttaagtgtaaaa 9822 9641 1 5 SEQ ID NO: 2898 tgattttcaagcaaatgca8536 8555 SEQ ID NO: 4236 tgcaagttaaagaaaatca 14025 14044 1 5 SEQ ID NO:2899 atgctgttttttggaaatg 8645 8664 SEQ ID NO: 4237 cattggtaggagacagcat11203 11222 1 5 SEQ ID NO: 2900 tgctgttttttggaaatgc 8646 8665 SEQ ID NO:4238 gcattggtaggagacagca 11202 11221 1 5 SEQ ID NO: 2901aaaaaaatacactggagct 8706 8725 SEQ ID NO: 4239 agctagagggcctcttttt 1083310652 1 5 SEQ ID NO: 2902 actggagcttagtaatgga 8716 8735 SEQ ID NO: 4240tccactcacatcctccagt 1289 1308 1 5 SEQ ID NO: 2903 cttctggaaaagggtcatg8886 8905 SEQ ID NO: 4241 catgaacccctacatgaag 13759 13778 1 5 SEQ ID NO:2904 ggaaaagggtcatggaaat 8891 8910 SEQ ID NO: 4242 atttgaaagttcgttttcc9282 9301 1 5 SEQ ID NO: 2905 gggcctgccccagattctc 8910 8929 SEQ ID NO:4243 gagaacattatggaggccc 9440 9459 1 5 SEQ ID NO: 2906ttctcagatgagggaacac 8924 8943 SEQ ID NO: 4244 gtgtcttcaaagctgagaa 1241612435 1 5 SEQ ID NO: 2907 gatgagggaacacatgaat 8930 8949 SEQ ID NO: 4245attccagcttccccacatc 8338 8357 1 5 SEQ ID NO: 2908 ctttggactgtccaataag8088 9005 SEQ ID NO: 4246 cttatgggatttcctaaag 11167 11186 1 5 SEQ ID NO:2909 gcatccacaaacaatgaag 9260 9279 SEQ ID NO: 4247 cttcatctgtcattgatgc10227 10246 1 5 SEQ ID NO: 2910 cacaaacaatgaagggaat 9265 9284 SEQ ID NO:4248 attccctgaagttgatgtg 11488 11507 1 5 SEQ ID NO: 2911ccaaaatttctctgctgga 9415 9434 SEQ ID NO: 4249 tccatcacaaatcctttgg 98719690 1 5 SEQ ID NO: 2912 caaaatttctctgctggaa 9416 9435 SEQ ID NO: 4250ttccatcacaaatcctttg 9670 9689 1 5 SEQ ID NO: 2913 tctgctggaaacaacgaga9425 9444 SEQ ID NO: 4251 tctcaagagttacagcaga 13229 13248 1 5 SEQ ID NO:2914 ctgctggaaacaacgagaa 9426 9445 SEQ ID NO: 4252 ttctcaagagttacagcag13228 13247 1 5 SEQ ID NO: 2915 agaacattatggaggccca 9441 9460 SEQ ID NO:4253 tgggcctgccccagattct 8909 8928 1 5 SEQ ID NO: 2916agaagcaaatctggatttc 9475 9494 SEQ ID NO: 4254 gaaatcttcaatttattct 1382113840 1 5 SEQ ID NO: 2917 tttctctctatgggaaaaa 9565 9584 SEQ ID NO: 4255tttttgcaagttaaagaaa 14021 14040 1 5 SEQ ID NO: 2918 tcagagcatcaaatccttt9712 9731 SEQ ID NO: 4256 aaagaaaatcaggatctga 14033 14052 1 5 SEQ ID NO:2919 cagaaacaatgcattagat 9751 9770 SEQ ID NO: 4257 atctatgccatctcttctg8663 5662 1 5 SEQ ID NO: 2920 tacacattaatcctgccat 10001 10020 SEQ ID NO:4258 atggagtctttattgtgta 14089 14108 1 5 SEQ ID NO: 2921agtcagatattgttgctca 10194 10213 SEQ ID NO: 4259 tgagaactacgagctgact 48074826 1 5 SEQ ID NO: 2922 ggagggtagtcataacagt 10336 10355 SEQ ID NO: 4260actggtggcaaaaccctcc 2734 2763 1 5 SEQ ID NO: 2923 caaaagccgaaattccaat10404 10423 SEQ ID NO: 4261 attgaagtacctacttttg 8366 8385 1 5 SEQ ID NO:2924 aaaagccgaaattccaatt 10405 10424 SEQ ID NO: 4262 aattgaagtacctactttt8655 8384 1 5 SEQ ID NO: 2925 ttcaagcaagaacttaatg 10436 10455 SEQ ID NO:4263 cattatggcccttcgtgaa 13258 13277 1 5 SEQ ID NO: 2926cctcttacttttccattga 10578 10597 SEQ ID NO: 4264 tcaaaagaagcccaagagg12947 12966 1 5 SEQ ID NO: 2927 tgaggccaacacttacttg 10663 10682 SEQ IDNO: 4265 caagcatctgattgactca 12678 12695 1 5 SEQ ID NO: 2928cacttacttgaattccaag 10672 10691 SEQ ID NO: 4266 cttgaacacaaagtcagtg 60086027 1 5 SEQ ID NO: 2929 gaagtaaaagaaaattttg 10751 10770 SEQ ID NO: 4267caaaaacattttcaacttc 5287 5306 1 5 SEQ ID NO: 2930 cctggaactctctccatgg10562 10901 SEQ ID NO: 4268 ccatttacagatcttcagg 11372 11391 1 5 SEQ IDNO: 2931 agctggatgtaaccaccag 11184 11203 SEQ ID NO: 4269ctggattccacatgcagct 11855 11874 1 5 SEQ ID NO: 2932 aaaattccctgaagttgat11485 11504 SEQ ID NO: 4270 atcatatccgtgtaatttt 6765 6784 1 5 SEQ ID NO:2933 cagatggcattgctgcttt 11813 11632 SEQ ID NO: 4271 aaagctgagaagaaatctg12424 12443 1 5 SEQ ID NO: 2934 agatggcattgctgctttg 11614 11833 SEQ IDNO: 4272 caaagctgagaagaaatct 12423 12442 1 5 SEQ ID NO: 2935tgttgaaacagtcctggat 11642 11861 SEQ ID NO: 4273 atccaagatgagatcaaca13103 13122 1 5 SEQ ID NO: 2936 catattcaaaactgagttg 12229 13248 SEQ IDNO: 4274 caactctctgattactatg 13631 13650 1 5 SEQ ID NO: 2937aaagatttatcaaaagaag 12938 12957 SEQ ID NO: 4275 cttcaatttattcttcttt13828 13845 1 5 SEQ ID NO: 2938 attttccaactaatagaag 13034 13053 SEQ IDNO: 4276 cttcaaagacttaaaaaat 8014 8033 1 5 SEQ ID NO: 2939aattatatccaagatgaga 13097 13116 SEQ ID NO: 4277 tctcttcctccatggaatt10479 10498 1 5 SEQ ID NO: 2940 ttcaggaagcttctcaaga 13218 13237 SEQ IDNO: 4278 tcttcataagttcaatgaa 13183 13202 1 5 SEQ ID NO: 2941ttgagcaatttctgcacag 13437 13456 SEQ ID NO: 4279 ctgttgaaagatttatcaa12932 12951 1 5 SEQ ID NO: 2942 ctgatatacatcacggagt 13712 13731 SEQ IDNO: 4280 actcaatggtgaaattcag 7465 7484 1 5 SEQ ID NO: 2943acatcacggagttactgaa 13719 13738 SEQ ID NO: 4281 ttcagaagctaagcaatgt 72397258 1 5 SEQ ID NO: 2944 actgcctatattgataaaa 13882 13901 SEQ ID NO: 4282ttttggcaagctatacagt 8380 8399 1 5 SEQ ID NO: 2945 aggatggcattttttgcaa14011 14030 SEQ ID NO: 4283 ttgcaagcaagtctttcct 3013 3032 1 5 SEQ ID NO:2946 ttttttgcaagttaaagaa 14020 14039 SEQ ID NO: 4284 ttctctctatgggaaaaaa9566 9585 1 5 SEQ ID NO: 2947 tccagaactcaagtcttca 1627 1646 SEQ ID NO:4285 tgaaatgctgttttttgga 8641 8660 3 4 SEQ ID NO: 2948agttagtgaaagaagttct 1956 1975 SEQ ID NO: 4286 agaatctgtaccaggaact 1256412583 3 4 SEQ ID NO: 2949 atttacagctctgacaagt 6435 5484 SEQ ID NO: 4287acttcagagaaatacaaat 11409 11428 3 4 SEQ ID NO: 2950 gattatctgaattcattca6488 6507 SEQ ID NO: 4288 tgaaaccaatgacaaaatc 7429 7448 3 4 SEQ ID NO:2951 gtgcccttctcggttgctg 26 45 SEQ ID NO: 4289 cagctgagcagacaggcac 60396058 2 4 SEQ ID NO: 2952 attcaagcacctccggaag 253 272 SEQ ID NO: 4290cttcataagttcaatgaat 13184 13203 2 4 SEQ ID NO: 2953 gactgctgattcaagaagt316 335 SEQ ID NO: 4291 acttcccaactctcaagtc 13415 13434 2 4 SEQ ID NO:2954 ttgctgcagccatgtccag 483 602 SEQ ID NO: 4292 ctgggcagctgtatagcaa5889 5908 2 4 SEQ ID NO: 2955 agaaagatgaacctactta 555 574 SEQ ID NO:4293 taagtatgatttcaattct 10498 10517 2 4 SEQ ID NO: 2956tgaagactctccaggaact 1095 1114 SEQ ID NO: 4294 agttcaatgaatttattca 1319113210 2 4 SEQ ID NO: 2957 atctctcttgccacagctg 1210 1229 SEQ ID NO: 4295cagcccagccatttgagat 9237 9256 2 4 SEQ ID NO: 2958 tctctcttgccacagctga1211 1230 SEQ ID NO: 4296 tcagcccagccatttgaga 9236 9256 2 4 SEQ ID NO:2959 tgaggtgtccagccccatc 1231 1250 SEQ ID NO: 4297 gatgggaaagccgccctca5218 5235 2 4 SEQ ID NO: 2960 ccagaactcaagtcttcaa 1628 1647 SEQ ID NO:4298 ttgaaagcagaacctctgg 5915 5934 2 4 SEQ ID NO: 2961ctgaaaaagttagtgaaag 1949 1968 SEQ ID NO: 4299 ctttctcgggaatattcag 1063110650 2 4 SEQ ID NO: 2962 tttttcccagacagtgtca 2246 2265 SEQ ID NO: 4300tgacaggcattttgaaaaa 9730 9749 2 4 SEQ ID NO: 2963 ttttcccagacagtgtcaa2247 2266 SEQ ID NO: 4301 ttgacaggcattttgaaaa 9729 9748 2 4 SEQ ID NO:2964 cattcagaacaagaaaatt 3403 3422 SEQ ID NO: 4302 aattccaattttgagaatg10414 10433 2 4 SEQ ID NO: 2965 tgaagagaagattgaattt 3628 3647 SEQ ID NO:4303 aaatgtcagctcttgttca 10902 10921 2 4 SEQ ID NO: 2966tttgaatggaacacaggcc 3644 3663 SEQ ID NO: 4304 tgccagtttgaaaaacaaa 1181511834 2 4 SEQ ID NO: 2967 ttctagattcgaatatcaa 4407 4426 SEQ ID NO: 4305ttgacatgttgataaagaa 7377 7396 2 4 SEQ ID NO: 2968 gattcgaatatcaaattca4412 4431 SEQ ID NO: 4306 tgaagtagaccaacaaatc 7162 7181 2 4 SEQ ID NO:2969 tgcaacgaccaacttgaag 5083 5102 SEQ ID NO: 4307 cttcaggttccatcgtgca11384 11403 2 4 SEQ ID NO: 2970 ttaagctctcaaatgacat 5325 5344 SEQ ID NO:4308 atgttgataaagaaattaa 7382 7401 2 4 SEQ ID NO: 2971caatttaacaacaatgaat 6074 6093 SEQ ID NO: 4309 attcaaactgcctatattg 1387613895 2 4 SEQ ID NO: 2972 tgaatacagccaggacttg 6088 6107 SEQ ID NO: 4310caagagcacacggtcttca 10587 10706 2 4 SEQ ID NO: 2973 catcaatattgatcaattt6421 6440 SEQ ID NO: 4311 aaattccctgaagttgatg 11486 11505 2 4 SEQ ID NO:2974 ttgagcatgtcaaacactt 7059 7078 SEQ ID NO: 4312 aagtaagtgctaggttcaa9381 9400 2 4 SEQ ID NO: 2975 tgaaggagactattcagaa 7227 7246 SEQ ID NO:4313 ttctgcacagaaatattca 13446 13465 2 4 SEQ ID NO: 2976ttcaggctcttcagaaagc 7929 7948 SEQ ID NO: 4314 gcttgctaacctctctgaa 1231212331 2 4 SEQ ID NO: 2977 tccacaaattgaacatccc 8787 8806 SEQ ID NO: 4315gggacctaccaagagtgga 12633 12552 2 4 SEQ ID NO: 2978 tgaataccaatgctgaact10167 10188 SEQ ID NO: 4316 agttcaatgaatttattca 13191 13210 2 4 SEQ IDNO: 2979 taaactaatagatgtaatc 12898 12917 SEQ ID NO: 4317gattactatgaaaaattta 13640 13669 2 4 SEQ ID NO: 2980 ttgacctgtccattcaaaa13680 13699 SEQ ID NO: 4318 ttttaaaagaaatcttcaa 13813 13832 2 4 SEQ IDNO: 2981 gggctgagtgcccttctcg 19 38 SEQ ID NO: 4319 cgaggccaggccgcagccc84 103 1 4 SEQ ID NO: 2982 ggctgagtgcccttctcgg 20 39 SEQ ID NO: 4320ccgaggccaggccgcagcc 83 102 1 4 SEQ ID NO: 2983 ctgagtgcccttctcggtt 22 41SEQ ID NO: 4321 aaccgtgcctgaatctcag 11657 11576 1 4 SEQ ID NO: 2984tctcggttgctgccgctga 33 52 SEQ ID NO: 4322 tcagctgacctcatcgaga 2168 21871 4 SEQ ID NO: 2985 caggccgcagcccaggagc 90 109 SEQ ID NO: 4323gctctgcagcttcatcctg 376 395 1 4 SEQ ID NO: 2986 gctggcgctgcctgcgctg 151170 SEQ ID NO: 4324 cagcacagaccatttcagc 4252 4271 1 4 SEQ ID NO: 2987tgctgctggcgggcgccag 177 196 SEQ ID NO: 4325 ctggatgtaaccaccagca 1118611205 1 4 SEQ ID NO: 2988 ctggtctgtccaaaagatg 227 246 SEQ ID NO: 4326catcctgaagaccagccag 388 407 1 4 SEQ ID NO: 2989 ctgagagttccagtggagt 291310 SEQ ID NO: 4327 actcaccctggacattcag 3391 3410 1 4 SEQ ID NO: 2990tccagtggagtccatggga 299 318 SEQ ID NO: 4328 tcccggagccaaggctgga 26832702 1 4 SEQ ID NO: 2991 aggttgagctggaggttcc 354 373 SEQ ID NO: 4329ggaaccctctccctcacct 4736 4755 1 4 SEQ ID NO: 2992 tgagctggaggttccccag358 377 SEQ ID NO: 4330 ctgggaggcatgatgctca 9171 9190 1 4 SEQ ID NO:2993 tctgcagcttcatcctgaa 378 397 SEQ ID NO: 4331 ttcaaatataatcggcaga3269 3288 1 4 SEQ ID NO: 2994 gccagtgcaccctgaaaga 402 421 SEQ ID NO:4332 tcttccgttctgtaatggc 5820 5821 1 4 SEQ ID NO: 2995ctctgaggagtttgctgca 472 491 SEQ ID NO: 4333 tgcaagaatattttgagag 63486367 1 4 SEQ ID NO: 2996 aggtatgagctcaagctgg 500 519 SEQ ID NO: 4334ccagtttccggggaaacct 12724 12743 1 4 SEQ ID NO: 2997 tcctttacccggagaaaga543 552 SEQ ID NO: 4335 tctttttgggaagcaagga 2227 2246 1 4 SEQ ID NO:2998 catcaagaggggcatcatt 583 602 SEQ ID NO: 4336 aatggtcaagttcctgatg2255 2304 1 4 SEQ ID NO: 2999 tcctggttcccccagagac 609 628 SEQ ID NO:4337 gtctctgaactcagaagga 13998 14015 1 4 SEQ ID NO: 3000aagaagccaagcaagtgtt 630 649 SEQ ID NO: 4338 aacaaataaatggagtctt 1408014099 1 4 SEQ ID NO: 3001 aagcaagtgttgtttctgg 638 657 SEQ ID NO: 4339ccagagccaggtcgagctt 11050 11069 1 4 SEQ ID NO: 3002 tctggataccgtgtatgga652 671 SEQ ID NO: 4340 tccatgtcccatttacaga 11364 11383 1 4 SEQ ID NO:3003 ccactcactttaccgtcaa 678 697 SEQ ID NO: 4341 ttgattttaacaaaagtgg6825 6844 1 4 SEQ ID NO: 3004 aggaagggcaatgtggcaa 701 720 SEQ ID NO:4342 ttgcaagcaagtctttcct 3013 3032 1 4 SEQ ID NO: 3005gcaatgtggcaacagaaat 708 727 SEQ ID NO: 4343 atttccataccccgtttgc 34833507 1 4 SEQ ID NO: 3006 caatgtggcaacagaaata 709 728 SEQ ID NO: 4344tattcttcttttccaattg 13834 13863 1 4 SEQ ID NO: 3007 tggcaacagaaatatccac714 733 SEQ ID NO: 4345 gtggcttcccatattgcca 1895 1914 1 4 SEQ ID NO:3008 agagacctgggccagtgtg 737 756 SEQ ID NO: 4346 cacattacatttggtctct2938 2957 1 4 SEQ ID NO: 3009 tgtgatcgcttcaagccca 752 771 SEQ ID NO:4349 tgggaaagccgccctcaca 5218 5237 1 4 SEQ ID NO: 3010gtgatcgcttcaagcccat 753 772 SEQ ID NO: 4350 atgggaaagccgccctcac 52175236 1 4 SEQ ID NO: 3011 cagcccacttgctctcatc 784 803 SEQ ID NO: 4351gatgctgaacagtgagctg 8152 8171 1 4 SEQ ID NO: 3012 gctctcatcaaaggcatga794 813 SEQ ID NO: 4352 tcataacagtactgtgagc 10346 10364 1 4 SEQ ID NO:3013 ccttgtcaactctgatcag 819 838 SEQ ID NO: 4353 ctgagtgggtttatcaagg12453 12472 1 4 SEQ ID NO: 3014 cttgtcaactctgatcagc 820 839 SEQ ID NO:4354 gctgagtgggtttatcaag 12452 12471 1 4 SEQ ID NO: 3015agccatctgcaaggagcaa 892 911 SEQ ID NO: 4355 ttgcaatgagctcatggct 38133832 1 4 SEQ ID NO: 3016 gccatctgcaaggagcaac 893 912 SEQ ID NO: 4356gttgcaatgagctcatggc 3812 3831 1 4 SEQ ID NO: 3017 cttcctgcctttctcctac916 935 SEQ ID NO: 4357 gtaggaataaatggagaag 9461 9480 1 4 SEQ ID NO:3018 ctttctcctacaagaataa 924 943 SEQ ID NO: 4358 ttattgctgaatccaaaag13866 13675 1 4 SEQ ID NO: 3019 gatcaacagccgcttcttt 997 1016 SEQ ID NO:4359 aaagccatcactgatgatc 1669 1688 1 4 SEQ ID NO: 3020atcaacagccgcttctttg 998 1017 SEQ ID NO: 4360 caaagccatcactgatgat 16681687 1 4 SEQ ID NO: 3021 acagccgcttctttggtga 1002 1021 SEQ ID NO: 4361tcacaaatcctttggctgt 9675 9694 1 4 SEQ ID NO: 3022 aagatgggcctcgcatttg1031 1060 SEQ ID NO: 4362 caaaatagaagggaatctt 2077 2096 1 4 SEQ ID NO:3023 tgttttgaagactctccag 1090 1109 SEQ ID NO: 4363 ctggtaactactttaaaca5495 5514 1 4 SEQ ID NO: 3024 ttgaagactctccaggaac 1094 1113 SEQ ID NO:4364 gttcaatgaatttattcaa 13192 13211 1 4 SEQ ID NO: 3025aactgaaaaaactaaccat 1110 1129 SEQ ID NO: 4365 atggcattttttgcaagtt 1401414033 1 4 SEQ ID NO: 3026 ctgaaaaaactaaccatct 1112 1131 SEQ ID NO: 4366agattgatgggcagttcag 4572 4691 1 4 SEQ ID NO: 3027 aaaactaaccatctctgag1117 1136 SEQ ID NO: 4367 ctcaaagaatgactttttt 2578 2597 1 4 SEQ ID NO:3028 tgagcaaaatatccagaga 1132 1151 SEQ ID NO: 4368 tctccagataaaaaactca12209 12228 1 4 SEQ ID NO: 3029 caataagctggttactgag 1162 1181 SEQ ID NO:4369 ctcagatcaaagttaattg 12273 12292 1 4 SEQ ID NO: 3030tactgagctgagaggccta 1174 1193 SEQ ID NO: 4370 gagggtagtcataacagta 1033710356 1 4 SEQ ID NO: 3031 gcctcagtgatgaagcagt 1188 1207 SEQ ID NO: 4371actgttgactcaggaaggc 12580 12599 1 4 SEQ ID NO: 3032 agtcacatctctcttgcca1204 1223 SEQ ID NO: 4372 tggccacatagcatggact 8866 8885 1 4 SEQ ID NO:3033 atctctcttgccacagctg 1210 1229 SEQ ID NO: 4373 cagctgacctcatcgagat2169 2188 1 4 SEQ ID NO: 3034 tctctcttgccacagctga 1211 1230 SEQ ID NO:4374 tcagctgacctcatcgaga 2168 2187 1 4 SEQ ID NO: 3035tgccacagctgattgaggt 1218 1237 SEQ ID NO: 4375 acctgcaccaaagctggca 1396313982 1 4 SEQ ID NO: 3036 gccacagctgattgaggtg 1219 1238 SEQ ID NO: 4376caccaaaaaccccaatggc 11248 11267 1 4 SEQ ID NO: 3037 tcactttacaagccttggt1248 1267 SEQ ID NO: 4377 accagatgctgaacagtga 8148 8167 1 4 SEQ ID NO:3038 cccttctgatagatgtggt 1332 1351 SEQ ID NO: 4378 accacttacagctagaggg10824 10843 1 4 SEQ ID NO: 3039 gtcacctacctggtggccc 1349 1368 SEQ ID NO:4379 gggcgacctaagttgtgac 3439 3458 1 4 SEQ ID NO: 3040ccttgtatgcgctgagcca 1440 1459 SEQ ID NO: 4380 tggctggtaacctaaaagg 55865605 1 4 SEQ ID NO: 3041 gacaaaccctacagggacc 1480 1499 SEQ ID NO: 4381ggtcctttatgattatgtc 12355 12374 1 4 SEQ ID NO: 3042 tgctaattacctgatggaa1516 1535 SEQ ID NO: 4382 ttcccaaaagcagtcagca 9938 9957 1 4 SEQ ID NO:3043 tgactgcactggggatgaa 1546 1565 SEQ ID NO: 4383 ttcaggtccatgcaagtca10917 10936 1 4 SEQ ID NO: 3044 actgcactggggatgaaga 1548 1567 SEQ ID NO:4384 tcttgaacacaaagtcagt 6007 6026 1 4 SEQ ID NO: 3045atgaagattacacctattt 1560 1579 SEQ ID NO: 4385 aaatgaaagtaaagatcat 81188137 1 4 SEQ ID NO: 3046 accatggagcagttaactc 1610 1628 SEQ ID NO: 4386gagtaaaccaaaacttggt 9024 9043 1 4 SEQ ID NO: 3047 gcagttaactccagaactc1618 1637 SEQ ID NO: 4387 gagttactgaaaaagctgc 13727 13746 1 4 SEQ ID NO:3048 cagaactcaagtcttcaat 1629 1648 SEQ ID NO: 4388 attggatatccaagatctg1933 1952 1 4 SEQ ID NO: 3049 caggctctgcggaaaatgg 1703 1722 SEQ ID NO:4389 ccatgacctccagctcctg 2485 2504 1 4 SEQ ID NO: 3050ccaggaggttcttcttcag 1738 1757 SEQ ID NO: 4390 ctgaaatacaatgctctgg 55195538 1 4 SEQ ID NO: 3051 ggttcttcttcagactttc 1744 1763 SEQ ID NO: 4391gaaaaacttggaaacaacc 4439 4458 1 4 SEQ ID NO: 3052 tttccttgatgatgcttct1759 1778 SEQ ID NO: 4392 agaatccagatacaagaaa 6893 6912 1 4 SEQ ID NO:3053 ggagataagcgactggctg 1781 1800 SEQ ID NO: 4393 cagcatgcctagtttctcc9952 9971 1 4 SEQ ID NO: 3054 gctgcctatcttatgttga 1796 1815 SEQ ID NO:4394 tcaatatcaaaagcccagc 12045 12064 1 4 SEQ ID NO: 3055actttgtggcttcccatat 1890 1909 SEQ ID NO: 4395 atatctggaaccttgaagt 1073710756 1 4 SEQ ID NO: 3056 gccaatatcttgaactcag 1910 1929 SEQ ID NO: 4396ctgaactcagaaggatggc 14000 14019 1 4 SEQ ID NO: 3057 aatatcttgaactcagaag1913 1932 SEQ ID NO: 4397 cttccattctgaatatatt 13378 13397 1 4 SEQ ID NO:3058 ctcagaagaattggatatc 1924 1943 SEQ ID NO: 4398 gataaaagattactttgag7273 7292 1 4 SEQ ID NO: 3059 aagaattggatatccaaga 1929 1943 SEQ ID NO:4399 tcttcaatttattcttctt 13825 13844 1 4 SEQ ID NO: 3060agaattggatatccaagat 1930 1949 SEQ ID NO: 4400 atcttcaatttattcttct 1382413843 1 4 SEQ ID NO: 3061 tggatatccaagatctgaa 1935 1954 SEQ ID NO: 4401ttcacataccagaattcca 8325 8344 1 4 SEQ ID NO: 3062 atatccaagatctgaaaaa1938 1957 SEQ ID NO: 4402 tttttaaccagtcagatat 10185 10204 1 4 SEQ ID NO:3063 tatccaagatctgaaaaag 1939 1958 SEQ ID NO: 4403 ctttttaaccagtcagata10184 10203 1 4 SEQ ID NO: 3064 caagatctgaaaaagttag 1943 1962 SEQ ID NO:4404 ctaaattcccatggtcttg 4973 4992 1 4 SEQ ID NO: 3065aagatctgaaaaagttagt 1944 1963 SEQ ID NO: 4405 actaaattcccatggtctt 49724991 1 4 SEQ ID NO: 3066 tgaaaaagttagtgaaaga 1950 1969 SEQ ID NO: 4406tctttctcgggaatattca 10630 10649 1 4 SEQ ID NO: 3067 tccaactgtcatggacttc1990 2009 SEQ ID NO: 4407 gaagcacatatgaactgga 13945 13964 1 4 SEQ ID NO:3068 tcagaaaattctctcggaa 2007 2026 SEQ ID NO: 4408 ttcctttaacaattcctga9501 9620 1 4 SEQ ID NO: 3069 ttccatcacttgacccagc 2052 2071 SEQ ID NO:4409 gctgacatagggaatggaa 8441 8460 1 4 SEQ ID NO: 3070cccagcctcagccaaaata 2065 2084 SEQ ID NO: 4410 tattctatccaagattggg 78207839 1 4 SEQ ID NO: 3071 agcctcagccaaaatagaa 2068 2087 SEQ ID NO: 4411ttctatccaagattgggct 7822 7841 1 4 SEQ ID NO: 3072 atcttatatttgatccaaa2091 2110 SEQ ID NO: 4412 tttgaaaaacaaagcagat 11821 11840 1 4 SEQ ID NO:3073 tcttatatttgatccaaat 2092 2111 SEQ ID NO: 4413 attttttgcaagttaaaga14019 14038 1 4 SEQ ID NO: 3074 cttcctaaagaaagcatgc 2117 2136 SEQ ID NO:4414 gcatggcattatgatgaag 3614 3633 1 4 SEQ ID NO: 3075ctaaagaaagcatgctgaa 2121 2140 SEQ ID NO: 4415 ttcagggtgtggagtttag 56945713 1 4 SEQ ID NO: 3076 taaagaaagcatgctgaaa 2122 2141 SEQ ID NO: 4416tttcttaaacattccttta 9490 9509 1 4 SEQ ID NO: 3077 gagattggcttggaaggaa2183 2202 SEQ ID NO: 4417 ttccctccattaagttctc 11709 11728 1 4 SEQ ID NO:3078 ctttgagccaacattggaa 2206 2225 SEQ ID NO: 4418 ttccaatgaccaagaaaag11068 11087 1 4 SEQ ID NO: 3079 cagacagtgtcaacaaagc 2253 2272 SEQ ID NO:4419 gcttactggacgaactctg 6142 6161 1 4 SEQ ID NO: 3080cagtgtcaacaaagctttg 2257 2278 SEQ ID NO: 4420 caaattcctggatacactg 98579876 1 4 SEQ ID NO: 3081 agtgtcaacaaagctttgt 2258 2277 SEQ ID NO: 4421acaagaatacgtctacact 4359 4378 1 4 SEQ ID NO: 3082 ctgatggtgtctctaaggt2298 2317 SEQ ID NO: 4422 acctcggaacaatcctcag 3333 3352 1 4 SEQ ID NO:3083 tgatggtgtctctaaggtc 2299 2318 SEQ ID NO: 4423 gacctgcgcaacgagatca8831 8860 1 4 SEQ ID NO: 3084 aaacatgagcaggatatgg 2351 2370 SEQ ID NO:4424 ccatgatctacatttgttt 6796 6815 1 4 SEQ ID NO: 3085gaagctgattaaagatttg 2395 2414 SEQ ID NO: 4425 caaaaacattttcaacttc 52875306 1 4 SEQ ID NO: 3086 aaagatttgaaatccaaag 2405 2424 SEQ ID NO: 4426ctttaagttcagcatcttt 7614 7633 1 4 SEQ ID NO: 3087 gatgggtgcccgcactctg2518 2537 SEQ ID NO: 4427 cagatttgaggattccatc 7983 8002 1 4 SEQ ID NO:3088 gggatcccccagatgattg 2540 2559 SEQ ID NO: 4428 caatcacaagtcgattccc9083 9102 1 4 SEQ ID NO: 3089 ttttcttcactacatcttc 2593 2612 SEQ ID NO:4429 gaagtgtcagtggcaaaaa 10382 10401 1 4 SEQ ID NO: 3090tcttcactacatcttcatg 2596 2615 SEQ ID NO: 4430 catggcattatgatgaaga 36153634 1 4 SEQ ID NO: 3091 tacatcttcatggagaatg 2603 2622 SEQ ID NO: 4431cattatggaggcccatgta 9445 9464 1 4 SEQ ID NO: 3092 ttcatggagaatgcctttg2609 2628 SEQ ID NO: 4432 caaaatcaactttaatgaa 6607 6626 1 4 SEQ ID NO:3093 tcatggagaatgcctttga 2610 2629 SEQ ID NO: 4433 tcaacacaatcttcaatga13116 13135 1 4 SEQ ID NO: 3094 tttgaactccccactggag 2624 2643 SEQ ID NO:4434 ctccccaggacctttcaaa 9842 9861 1 4 SEQ ID NO: 3095ttgaactccccactggagc 2625 2644 SEQ ID NO: 4435 gctccccaggacctttcaa 98419880 1 4 SEQ ID NO: 3096 tgaactccccactggagct 2626 2645 SEQ ID NO: 4436agctccccaggacctttca 9840 9859 1 4 SEQ ID NO: 3097 cactggagctggattacag2635 2654 SEQ ID NO: 4437 ctgtttctgagtcccagtg 9344 9363 1 4 SEQ ID NO:3098 actggagctggattacagt 2636 2655 SEQ ID NO: 4438 actgtttctgagtcccagt9343 9352 1 4 SEQ ID NO: 3099 agttgcaaatatcttcatc 2652 2671 SEQ ID NO:4439 gatgatgccaaaatcaact 6599 6618 1 4 SEQ ID NO: 3100gttgcaaatatcttcatct 2653 2672 SEQ ID NO: 4440 agatgatgccaaaatcaac 65986617 1 4 SEQ ID NO: 3101 aaatatcttcatctggagt 2658 2677 SEQ ID NO: 4441actcagaaggatggcattt 14004 14023 1 4 SEQ ID NO: 3102 taaaactggaagtagccaa2703 2722 SEQ ID NO: 4442 ttggttacaggaggcttta 7600 7619 1 4 SEQ ID NO:3103 ggctgaactggtggcaaaa 2728 2747 SEQ ID NO: 4443 ttttcttttcagcccagcc9228 9247 1 4 SEQ ID NO: 3104 tgtggagtttgtgacaaat 2758 2777 SEQ ID NO:4444 attttcaagcaaatgcaca 8538 8557 1 4 SEQ ID NO: 3105ttgtgacaaatatgggcat 2766 2785 SEQ ID NO: 4445 atgcgtctaccttacacaa 85219540 1 4 SEQ ID NO: 3106 atgaacaccaacttcttcc 2819 2838 SEQ ID NO: 4446ggaagctgaagtttatcat 2877 2896 1 4 SEQ ID NO: 3107 cttccacgagtcgggtctg2833 2852 SEQ ID NO: 4447 cagagctatcactgggaag 5235 5254 1 4 SEQ ID NO:3108 gagtcgggtctggaggctc 2840 2859 SEQ ID NO: 4448 gagcttactggacgaactc6140 6159 1 4 SEQ ID NO: 3109 cctaaaagctgggaagctg 2866 2885 SEQ ID NO:4449 cagcctccccagccgtagg 12120 12139 1 4 SEQ ID NO: 3110agctgggaagctgaagttt 2872 2891 SEQ ID NO: 4450 aaactgttaatttacagct 54635482 1 4 SEQ ID NO: 3111 ccagattagagctggaact 3114 3133 SEQ ID NO: 4451agtttccggggaaacctgg 12726 12745 1 4 SEQ ID NO: 3112 ggataccctgaagtttgta3208 3227 SEQ ID NO: 4452 tacagtattctgaaaatcc 8393 8412 1 4 SEQ ID NO:3113 ctgaggctaccatgacatt 3252 3271 SEQ ID NO: 4453 aatgagctcatggcttcag3817 3836 1 4 SEQ ID NO: 3114 tgtccagtgaagtccaaat 3297 3316 SEQ ID NO:4454 attttgagaggaatcgaca 6357 6376 1 4 SEQ ID NO: 3115aattccggattttgatgtt 3313 3332 SEQ ID NO: 4455 aacacatgaatcacaaatt 89388957 1 4 SEQ ID NO: 3116 ttccggattttgatgttga 3315 3334 SEQ ID NO: 4456tcaaaacgagcttcaggaa 13207 13226 1 4 SEQ ID NO: 3117 cggaacaatcctcagagtt3337 3356 SEQ ID NO: 4457 aacttgtacaactggtccg 4211 4230 1 4 SEQ ID NO:3118 tcctcagagttaatgatga 3345 3364 SEQ ID NO: 4458 tcatcaattggttacagga7593 7612 1 4 SEQ ID NO: 3119 ctcaccctggacattcaga 3392 3411 SEQ ID NO:4459 tctgcagaacaatgctgag 12439 12458 1 4 SEQ ID NO: 3120cattcagaacaagaaaatt 3403 3422 SEQ ID NO: 4460 aattgactttgtagaaatg 81048123 1 4 SEQ ID NO: 3121 actgaggtcgccctcatgg 3422 3441 SEQ ID NO: 4461ccatgcaagtcagcccagt 10924 10943 1 4 SEQ ID NO: 3122 ttatttccataccccgttt3486 3505 SEQ ID NO: 4462 aaactgcctatattgataa 13880 13899 1 4 SEQ ID NO:3123 gtttgcaagcagaagccag 3501 3520 SEQ ID NO: 4463 ctggacttctcttcaaaac5408 5427 1 4 SEQ ID NO: 3124 tttgcaagcagaagccaga 3502 3521 SEQ ID NO:4464 tctgggtgtcgacagcaaa 5272 5291 1 4 SEQ ID NO: 3125ttgcaagcagaagccagaa 3503 3522 SEQ ID NO: 4465 ttctgggtgtcgacagcaa 52715290 1 4 SEQ ID NO: 3126 ctgcttctccaaatggact 3554 3573 SEQ ID NO: 4466agtcaagattgatgggcag 4567 4586 1 4 SEQ ID NO: 3127 tgctacagcttatggctcc3577 3598 SEQ ID NO: 4467 ggaggctttaagttcagca 7809 7628 1 4 SEQ ID NO:3128 acagcttatggctccacag 3581 3600 SEQ ID NO: 4468 ctgtatagcaaattcctgt5897 5916 1 4 SEQ ID NO: 3129 tttccaagagggtggcatg 3600 3619 SEQ ID NO:4469 catggacttcttctggaaa 8877 8896 1 4 SEQ ID NO: 3130ccaagagggtggcatggca 3603 3622 SEQ ID NO: 4470 tgcccagcaagcaagttgg 93619380 1 4 SEQ ID NO: 3131 gtggcatggcattatgatg 3611 3630 SEQ ID NO: 4471catccttaacaccttccac 8071 8090 1 4 SEQ ID NO: 3132 tgatgaagagaagattgaa3625 3644 SEQ ID NO: 4472 ttcactgttcctgaaatca 7871 7890 1 4 SEQ ID NO:3133 gaagagaagattgaatttg 3629 3648 SEQ ID NO: 4473 caaaaacattttcaacttc5287 5306 1 4 SEQ ID NO: 3134 gagaagattgaatttgaat 3632 3651 SEQ ID NO:4474 attcataatcccaactctc 8275 8297 1 4 SEQ ID NO: 3135tttgaatggaacacaggca 3644 3663 SEQ ID NO: 4475 tgcctttgtgtacaccaaa 1123611255 1 4 SEQ ID NO: 3136 aggcaccaatgtagatacc 3658 3677 SEQ ID NO: 4476ggtaacctaaaaggagcct 5591 5810 1 4 SEQ ID NO: 3137 caaaaaaatgacttccaat3676 3695 SEQ ID NO: 4477 attgaagtacctacttttg 8366 8385 1 4 SEQ ID NO:3138 aaaaaaatgacttccaatt 3677 3696 SEQ ID NO: 4478 aattgaagtacctactttt8365 8384 1 4 SEQ ID NO: 3139 aaaaaatgacttccaattt 3678 3697 SEQ ID NO:4479 aaatccaatctcctctttt 8408 8425 1 4 SEQ ID NO: 3140cagagtccctcaaacagac 3760 3779 SEQ ID NO: 4480 gtctgtgggattccatctg 40904109 1 4 SEQ ID NO: 3141 aaattaatagttgcaatga 3803 3822 SEQ ID NO: 4481tcataagttcaatgaattt 13188 13205 1 4 SEQ ID NO: 3142 ttcaacctccagaacatgg3809 3918 SEQ ID NO: 4482 ccattgaccagatgctgaa 8142 8161 1 4 SEQ ID NO:3143 tgggattgccagacttcca 3915 3934 SEQ ID NO: 4483 tggaaatgggcctgcccca8903 8922 1 4 SEQ ID NO: 3144 cagtttgaaaattgagatt 3994 4013 SEQ ID NO:4484 aatcacaactcctccactg 9541 9560 1 4 SEQ ID NO: 3145gaaaattgagattcctttg 4000 4019 SEQ ID NO: 4485 caaaactaccacacatttc 1369413713 1 4 SEQ ID NO: 3146 tttgccttttggtggcaaa 4015 4034 SEQ ID NO: 4486tttgagaggaatcgacaaa 6359 6378 1 4 SEQ ID NO: 3147 ctccagagatctaaagatg4036 4055 SEQ ID NO: 4487 catcaattggttacaggag 7594 7613 1 4 SEQ ID NO:3148 tctaaagatgttagagact 4045 4064 SEQ ID NO: 4488 agtccttcatgtccctaga10033 10052 1 4 SEQ ID NO: 3149 ctgtgggattccatctgcc 4092 4111 SEQ ID NO:4489 ggcattttgaaaaaaacag 9735 9754 1 4 SEQ ID NO: 3150atctgccatctcgagagtt 4104 4123 SEQ ID NO: 4490 aactctcaaaccctaagat 85568575 1 4 SEQ ID NO: 3151 tctcgagagttccaagtcc 4112 4131 SEQ ID NO: 4491ggacattcctctagcgaga 8215 8234 1 4 SEQ ID NO: 3152 agtccctacttttaccatt4126 4145 SEQ ID NO: 4492 aatgaatacagccaggact 6086 6105 1 4 SEQ ID NO:3153 acttttaccattcccaagt 4133 4152 SEQ ID NO: 4493 actttgtagaaatgaaagt8109 8128 1 4 SEQ ID NO: 3154 cattcccaagttgtatcaa 4141 4160 SEQ ID NO:4494 ttgaaggacttcaggaatg 12009 12028 1 4 SEQ ID NO: 3155accacatgaaggctgactc 4284 4303 SEQ ID NO: 4495 gagtaaaccaaaacttggt 90249043 1 4 SEQ ID NO: 3156 tttcctacaatgtgcaagg 4317 4336 SEQ ID NO: 4496cctttaacaattcctgaaa 9503 9522 1 4 SEQ ID NO: 3157 ctggagaaacaacatatga4338 4357 SEQ ID NO: 4497 tcattctgggtctttccag 11035 11054 1 4 SEQ ID NO:3158 atcatgtgatgggtctcta 4378 4397 SEQ ID NO: 4498 tagaattacagaaaatgat6555 6584 1 4 SEQ ID NO: 3159 catgtgatgggtctctacg 4380 4399 SEQ ID NO:4499 cgtaggcaccgtgggcatg 12133 12182 1 4 SEQ ID NO: 3160ttctagattcgaatatcaa 4407 4428 SEQ ID NO: 4500 ttgatgatgctgtcaagaa 73087327 1 4 SEQ ID NO: 3161 tggggaccacagatgtctg 4499 4516 SEQ ID NO: 4501cagaattccagcttcccca 8334 8353 1 4 SEQ ID NO: 3162 ctaacactggccggctcaa4644 4663 SEQ ID NO: 4502 ttgaggctattgatgttag 6984 7003 1 4 SEQ ID NO:3163 taacactggccggctcaat 4645 4664 SEQ ID NO: 4503 attgaggctattgatgtta6983 7002 1 4 SEQ ID NO: 3164 aacactggccggctcaatg 4646 4665 SEQ ID NO:4504 cattgaggctattgatgtt 6982 7001 1 4 SEQ ID NO: 3165ctggccggctcaatggaga 4650 4669 SEQ ID NO: 4505 tctccatctgcgctaccag 1207312092 1 4 SEQ ID NO: 3166 agataacaggaagatatga 4713 4732 SEQ ID NO: 4506tcatctcctttcttcatct 10210 10229 1 4 SEQ ID NO: 3167 tccctcacctccacctctg4745 4764 SEQ ID NO: 4507 cagatatatatctcaggga 8184 8203 1 4 SEQ ID NO:3168 agctgactttaaaatctga 4818 4837 SEQ ID NO: 4508 tcaggctcttcagaaagct7930 7949 1 4 SEQ ID NO: 3169 ctgactttaaaatctgaca 4820 4839 SEQ ID NO:4509 tgtcaagataaacaatcag 8740 8759 1 4 SEQ ID NO: 3170caagatggatatgaccttc 4873 4892 SEQ ID NO: 4510 gaagtagtactgcatcttg 88436862 1 4 SEQ ID NO: 3171 gctgcgttctgaatatcag 4909 4928 SEQ ID NO: 4511ctgagtcccagtgcccagc 8350 9369 1 4 SEQ ID NO: 3172 cgttctgaatatcaggctg4913 4932 SEQ ID NO: 4512 cagcaagtacctgagaacg 8611 8830 1 4 SEQ ID NO:3173 aattcccatggtcttgagt 4976 4995 SEQ ID NO: 4513 actcagatcaaagttaatt12272 12291 1 4 SEQ ID NO: 3174 tggtcttgagttaaatgct 4984 5003 SEQ ID NO:4514 agcacagtacgaaaaacca 10809 10828 1 4 SEQ ID NO: 3175cttgagttaaatgctgaca 4988 5007 SEQ ID NO: 4515 tgtccctagaaatctcaag 1004210061 1 4 SEQ ID NO: 3176 ttgagttaaatgctgacat 4989 5008 SEQ ID NO: 4516atgtccctagaaatctcaa 10041 10060 1 4 SEQ ID NO: 3177 tgagttaaatgctgacatc4990 5009 SEQ ID NO: 4517 gatggaaccctctccctca 4733 4752 1 4 SEQ ID NO:3178 acttgaagtgtagtctcct 5094 5113 SEQ ID NO: 4518 aggaaactcagatcaaagt12267 12286 1 4 SEQ ID NO: 3179 agtgtagtctcctggtgct 5100 5119 SEQ ID NO:4519 agcagccagtggcaccact 12514 12633 1 4 SEQ ID NO: 3180gtgctggagaatgagctga 5114 5133 SEQ ID NO: 4520 tcagccaggtttatagcac 77347753 1 4 SEQ ID NO: 3181 ctggggcatctatgaaatt 5151 5170 SEQ ID NO: 4521aatttctgattaccaccag 13579 13598 1 4 SEQ ID NO: 3182 atggccgcttcagggaaca5178 5197 SEQ ID NO: 4522 tgttttttggaaatgccat 8649 8668 1 4 SEQ ID NO:3183 ttcagtctggatgggaaag 5207 5226 SEQ ID NO: 4523 ctttgacaggcattttgaa9727 9746 1 4 SEQ ID NO: 3184 ccatgattctgggtgtcga 5265 5284 SEQ ID NO:4524 tcgatgcacatacaaatgg 5838 5857 1 4 SEQ ID NO: 3185aaaacattttcaacttcaa 5289 5308 SEQ ID NO: 4525 ttgatgttagagtgctttt 69937012 1 4 SEQ ID NO: 3186 cttaagctctcaaatgaca 5324 5343 SEQ ID NO: 4526tgtcctacaacaagttaag 7255 7274 1 4 SEQ ID NO: 3187 ttaagctctcaaatgacat5325 5344 SEQ ID NO: 4527 atgtcctacaacaagttaa 7254 7273 1 4 SEQ ID NO:3188 catgatgggctcatatgct 5341 5360 SEQ ID NO: 4528 agcatctttggctcacatg7624 7643 1 4 SEQ ID NO: 3189 tgggctcatatgctgaaat 5346 5365 SEQ ID NO:4529 atttatcaaaagaagccca 12942 12961 1 4 SEQ ID NO: 3190actggacttctcttcaaaa 5407 5426 SEQ ID NO: 4530 ttttggcaagctatacagt 63808399 1 4 SEQ ID NO: 3191 acttctcttcaaaacttga 5412 5431 SEQ ID NO: 4531tcaattgggagagacaagt 6504 6523 1 4 SEQ ID NO: 3192 ctgacaagttttataagca5445 5464 SEQ ID NO: 4532 tgctttgtgagtttatcag 9693 9712 1 4 SEQ ID NO:3193 aagttttataagcaaactg 5450 5469 SEQ ID NO: 4533 cagtcatgtagaaaaactt4429 4448 1 4 SEQ ID NO: 3194 ctgttaatttacagctaca 5466 5485 SEQ ID NO:4534 tgtactggaaaacgtacag 6388 6407 1 4 SEQ ID NO: 3195ttacagctacagccctatt 5474 5493 SEQ ID NO: 4535 aatattgatcaatttgtaa 64256444 1 4 SEQ ID NO: 3196 tctggtaactactttaaac 5494 5513 SEQ ID NO: 4536gtttgaaaaacaaagcaga 11820 11839 1 4 SEQ ID NO: 3197 tttaaacagtgacctgaaa5506 5525 SEQ ID NO: 4537 tttcatttgaaagaataaa 7032 7051 1 4 SEQ ID NO:3198 ttaaacagtgacctgaaat 5507 5526 SEQ ID NO: 4538 atttcaagcaagaacttaa10434 10453 1 4 SEQ ID NO: 3199 cagtgacctgaaatacaat 5512 5531 SEQ ID NO:4539 attggcgtggagcttactg 6131 6150 1 4 SEQ ID NO: 3200tgtggctggtaacctaaaa 5584 5603 SEQ ID NO: 4540 ttttgctggagaagccaca 1076510784 1 4 SEQ ID NO: 3201 ttatcagcaagctataaag 5657 5676 SEQ ID NO: 4541ctttgcactatgttcataa 12764 12783 1 4 SEQ ID NO: 3202 ggttcagggtgtggagttt5692 5711 SEQ ID NO: 4542 aaacacctaagagtaaacc 9014 8033 1 4 SEQ ID NO:3203 attcagactcactgcattt 5775 5794 SEQ ID NO: 4543 aaatgctgacatagggaat8437 8456 1 4 SEQ ID NO: 3204 ttcagactcactgcatttc 5776 5795 SEQ ID NO:4544 gaaatattatgaacttgaa 13312 13331 1 4 SEQ ID NO: 3205tacaaatggcaatgggaaa 5848 5867 SEQ ID NO: 4545 tttcctaaagctggatgta 1117611195 1 4 SEQ ID NO: 3206 gctgtatagcaaattcctg 5886 5915 SEQ ID NO: 4546caggtccatgcaagtcagc 10919 10938 1 4 SEQ ID NO: 3207 tgagcagacaggcacctgg6043 6062 SEQ ID NO: 4547 ccagcttccccacatctca 8341 8360 1 4 SEQ ID NO:3208 ggcacctggaaactcaaga 6053 6072 SEQ ID NO: 4548 tcttcgtgtttcaactgcc11221 11240 1 4 SEQ ID NO: 3209 tgaatacagccaggacttg 6088 6107 SEQ ID NO:4549 caagtaagtgctaggttca 9380 9399 1 4 SEQ ID NO: 3210gaatacagccaggacttgg 6089 6108 SEQ ID NO: 4550 ccaacacttacttgaattc 1066810687 1 4 SEQ ID NO: 3211 ctggacgaactctggctga 6147 6166 SEQ ID NO: 4551tcagaaagctaccttccag 7939 7958 1 4 SEQ ID NO: 3212 ttttactcagtgagcccat6201 6220 SEQ ID NO: 4552 atggacttcttctggaaaa 8878 8897 1 4 SEQ ID NO:3213 gatgagagatgccgttgag 6241 6260 SEQ ID NO: 4553 ctcatctcctttcttcatc10209 10228 1 4 SEQ ID NO: 3214 aattgttgcttttgtaaag 6277 6296 SEQ ID NO:4554 cttttctaaacttgaaatt 9064 9083 1 4 SEQ ID NO: 3215cttttgtaaagtatgataa 6285 6304 SEQ ID NO: 4555 ttatgaacttgaagaaaag 1331813337 1 4 SEQ ID NO: 3216 tttgtaaagtatgataaaa 6287 6306 SEQ ID NO: 4556ttttcacattagatgcaaa 8421 8440 1 4 SEQ ID NO: 3217 tccattaacctcccatttt6320 6339 SEQ ID NO: 4557 aaaattgatgatatctgga 10727 10746 1 4 SEQ ID NO:3218 ccattaacctcccattttt 6321 6340 SEQ ID NO: 4558 aaaagggtcatggaaatgg8893 8912 1 4 SEQ ID NO: 3219 cttgcaagaatattttgag 6346 6365 SEQ ID NO:4559 ctcaattttgattttcaag 8528 8547 1 4 SEQ ID NO: 3220agaatattttgagaggaat 6352 6371 SEQ ID NO: 4560 attccctccattaagttct 1170811727 1 4 SEQ ID NO: 3221 attatagttgtactggaaa 6380 6399 SEQ ID NO: 4561tttcaagcaagaacttaat 10435 10454 1 4 SEQ ID NO: 3222 gaagcacatcaatattgat6415 6434 SEQ ID NO: 4562 atcagttcagataaacttc 7999 8018 1 4 SEQ ID NO:3223 acatcaatattgatcaatt 6420 6439 SEQ ID NO: 4563 aattccctgaagttgatgt11487 11506 1 4 SEQ ID NO: 3224 gaaaactcccacagcaagc 6465 6484 SEQ ID NO:4564 gctttctcttccacatttc 10060 10079 1 4 SEQ ID NO: 3225ctgaattcattcaattggg 6494 6513 SEQ ID NO: 4565 cccatttacagatcttcag 1137111390 1 4 SEQ ID NO: 3226 tgaattcattcaattggga 6495 6514 SEQ ID NO: 4566tcccatttacagatcttca 11370 11389 1 4 SEQ ID NO: 3227 aactgactgctctcacaaa6540 6559 SEQ ID NO: 4567 tttgaggattccatcagtt 7987 8006 1 4 SEQ ID NO:3228 aaaagtatagaattacaga 6558 6577 SEQ ID NO: 4568 tctggctccctcaactttt9050 9069 1 4 SEQ ID NO: 3229 atcaactttaatgaaaaac 6611 6630 SEQ ID NO:4569 gtttattgaaaatattgat 6811 6830 1 4 SEQ ID NO: 3230tgatttgaaaatagctatt 6694 6713 SEQ ID NO: 4570 aatattattgatgaaatca 67156735 1 4 SEQ ID NO: 3231 atttgaaaatagctattgc 6696 6715 SEQ ID NO: 4571gcaagaacttaatggaaat 10441 10460 1 4 SEQ ID NO: 3232 attgctaatattattgatg6710 6729 SEQ ID NO: 4572 catcacactgaataccaat 10159 10178 1 4 SEQ ID NO:3233 gaaaaattaaaaagtcttg 6737 6756 SEQ ID NO: 4573 caagagcttatgggatttc11161 11180 1 4 SEQ ID NO: 3234 actatcatatccgtgtaat 6762 6781 SEQ ID NO:4574 attactttgagaaattagt 7281 7300 1 4 SEQ ID NO: 3235tattgattttaacaaaagt 6823 6842 SEQ ID NO: 4575 acttgacttcagagaaata 1140411423 1 4 SEQ ID NO: 3236 ctgcagcagcttaagagac 6914 6933 SEQ ID NO: 4576gtcttcagtgaagctgcag 10699 10718 1 4 SEQ ID NO: 3237 aaaacaacacattgaggct6973 6992 SEQ ID NO: 4577 agcctcacctcttactttt 10571 10590 1 4 SEQ ID NO:3238 ttgagcatgtcaaacactt 7059 7078 SEQ ID NO: 4578 aagtagctgagaaaatcaa7104 7123 1 4 SEQ ID NO: 3239 tttgaagtagctgagaaaa 7100 7119 SEQ ID NO:4579 ttttcacattagatgcaaa 8421 8440 1 4 SEQ ID NO: 3240ttagtagagttggcccacc 7199 7218 SEQ ID NO: 4580 ggtggactcttgctgctaa 77767795 1 4 SEQ ID NO: 3241 tgaaggagactattcagaa 7227 7246 SEQ ID NO: 4581ttctcaattttgattttca 8526 8545 1 4 SEQ ID NO: 3242 gagactattcagaagctaa7232 7251 SEQ ID NO: 4582 ttagccacagctctgtctc 10301 10320 1 4 SEQ ID NO:3243 aattagttggatttattga 7293 7312 SEQ ID NO: 4583 tcaagaagcttaatgaatt7320 7339 1 4 SEQ ID NO: 3244 gcttaatgaattatctttt 7327 7346 SEQ ID NO:4584 aaaacgagcttcaggaagc 13209 13228 1 4 SEQ ID NO: 3245ttaacaaattccttgacat 7365 7384 SEQ ID NO: 4585 atgtcctacaacaagttaa 72547273 1 4 SEQ ID NO: 3246 aaattaaagtcatttgatt 7394 7413 SEQ ID NO: 4586aatcctttgacaggcattt 9723 9742 1 4 SEQ ID NO: 3247 gactcaatggtgaaattca7464 7488 SEQ ID NO: 4587 tgaaattcaatcacaagtc 9076 9095 1 4 SEQ ID NO:3248 gaaattcaggctctggaac 7475 7494 SEQ ID NO: 4588 gttctcaattttgattttc8525 8544 1 4 SEQ ID NO: 3249 actaccacaaaaagctgaa 7492 7511 SEQ ID NO:4589 ttcaggaactattgctagt 10645 10664 1 4 SEQ ID NO: 3250ccaaaataaccttaatcat 7578 7597 SEQ ID NO: 4590 atgatttccctgaccttgg 1095010969 1 4 SEQ ID NO: 3251 aaataaccttaatcatcaa 7581 7600 SEQ ID NO: 4591ttgaagtaaaagaaaattt 10749 10768 1 4 SEQ ID NO: 3252 tttaagttcagcatctttg7615 7634 SEQ ID NO: 4592 caaatctggatttcttaaa 9480 9499 1 4 SEQ ID NO:3253 caggtttatagcacacttg 7739 7758 SEQ ID NO: 4593 caagggttcactgttcctg7865 7884 1 4 SEQ ID NO: 3254 gttcactgttcctgaaatc 7870 7889 SEQ ID NO:4594 gattctcagatgagggaac 8922 8941 1 4 SEQ ID NO: 3255cactgttcctgaaatcaag 7873 7892 SEQ ID NO: 4595 cttgaacacaaagtcagtg 60086027 1 4 SEQ ID NO: 3256 actgttcctgaaatcaaga 7874 7893 SEQ ID NO: 4596tcttgaacacaaagtcagt 6007 6026 1 4 SEQ ID NO: 3257 gcctgcctttgaagtcagt7909 7928 SEQ ID NO: 4597 actgttgactcaggaaggc 12580 12599 1 4 SEQ ID NO:3258 taacagatttgaggattcc 7980 7999 SEQ ID NO: 4598 ggaagcttctcaagagtta13222 13241 1 4 SEQ ID NO: 3259 gttttccacaccagaattt 8050 8069 SEQ ID NO:4599 aaatttctctgctggaaac 9418 9437 1 4 SEQ ID NO: 3260tcagaaccattgaccagat 8136 8155 SEQ ID NO: 4600 atctgcagaacaatgctga 1243812457 1 4 SEQ ID NO: 3261 tagcgagaatcaccctgcc 8226 8245 SEQ ID NO: 4601ggcagcttctggcttgcta 12301 12320 1 4 SEQ ID NO: 3262 ccttaatgattttcaagtt8299 8318 SEQ ID NO: 4602 aactgttgactcaggaagg 12579 12598 1 4 SEQ ID NO:3263 acataccagaattccagct 8328 8347 SEQ ID NO: 4603 agctgccagtccttcatgt10026 10045 1 4 SEQ ID NO: 3264 aatgctgacatagggaatg 8438 8457 SEQ ID NO:4604 cattaatcctgccatcatt 10005 10024 1 4 SEQ ID NO: 3265atgctgacatagggaatgg 8439 8458 SEQ ID NO: 4605 ccatttgagatcacggcat 92459264 1 4 SEQ ID NO: 3266 aaccacctcagcaaacgaa 8458 8477 SEQ ID NO: 4606ttcgttttccattaaggtt 9291 9310 1 4 SEQ ID NO: 3267 agcaggtatcgcagcttcc8476 8495 SEQ ID NO: 4607 ggaagtggccctgaatgct 10972 10991 1 4 SEQ ID NO:3268 tgcacaactctcaaaccct 8551 8570 SEQ ID NO: 4608 agggaaagagaagattgca13501 13520 1 4 SEQ ID NO: 3269 aggagtcagtgaagttctc 8592 8611 SEQ ID NO:4609 gagaacttactatcatcct 13788 13807 1 4 SEQ ID NO: 3270tttttggaaatgccattga 8652 8671 SEQ ID NO: 4610 tcaatgaatttattcaaaa 1319413213 1 4 SEQ ID NO: 3271 aatggagtgattgtcaaga 8729 8748 SEQ ID NO: 4611tcttttcagcccagccatt 9231 9250 1 4 SEQ ID NO: 3272 gtcaagataaacaatcagc8741 8760 SEQ ID NO: 4612 gctgactttaaaatctgac 4819 4838 1 4 SEQ ID NO:3273 tccacaaattgaacatccc 8787 8806 SEQ ID NO: 4613 gggatttcctaaagctgga11172 11191 1 4 SEQ ID NO: 3274 ttgaacatccccaaactgg 8795 8814 SEQ ID NO:4614 ccagtttccagggactcaa 12603 12622 1 4 SEQ ID NO: 3275acatccccaaactggactt 8799 8818 SEQ ID NO: 4615 aagtcgattcccagcatgt 90909109 1 4 SEQ ID NO: 3276 acttctctagtcaggctga 8814 8833 SEQ ID NO: 4616tcagatggaaaaatgaagt 11010 11029 1 4 SEQ ID NO: 3277 tgaatcacaaattagtttc8944 8963 SEQ ID NO: 4617 gaaagtccataatggttca 12817 12836 1 4 SEQ ID NO:3278 agaaggacccctcacttcc 8968 8987 SEQ ID NO: 4618 ggaagaagaggcagcttct12292 12311 1 4 SEQ ID NO: 3279 ttggactgtccaataagat 8988 9007 SEQ ID NO:4619 atctaaatgcagtagccaa 11634 11653 1 4 SEQ ID NO: 3280actgtccaataagatcaat 8992 9011 SEQ ID NO: 4620 attgataaaaccatacagt 1389113910 1 4 SEQ ID NO: 3281 ctgtccaataagatcaata 8993 9012 SEQ ID NO: 4621tattgataaaaccatacag 13890 13909 1 4 SEQ ID NO: 3282 gtttatgaatctggctccc9041 9060 SEQ ID NO: 4622 gggaatctgatgaggaaac 12255 12274 1 4 SEQ ID NO:3283 atgaatctggctccctcaa 9045 9064 SEQ ID NO: 4623 ttgagttgcccaccatcat11667 11686 1 4 SEQ ID NO: 3284 ctcaacttttctaaacttg 9059 9078 SEQ ID NO:4624 caagatcgcagactttgag 11653 11672 1 4 SEQ ID NO: 3285ctaaaggcatggcactgtt 9129 9148 SEQ ID NO: 4625 aacagaaacaatgcattag 97499768 1 4 SEQ ID NO: 3286 aaggcatggcactgtttgg 9132 9151 SEQ ID NO: 4626ccaagaaaaggcacacctt 11077 11096 1 4 SEQ ID NO: 3287 atccacaaacaatgaaggg9262 9281 SEQ ID NO: 4627 ccctaacagatttgaggat 7977 7996 1 4 SEQ ID NO:3288 ggaatttgaaagttcgttt 9279 9298 SEQ ID NO: 4628 aaacaaacacaggcattcc9655 9674 1 4 SEQ ID NO: 3289 aataactatgcactgtttc 9332 9351 SEQ ID NO:4629 gaaatactgttttcctatt 12836 12855 1 4 SEQ ID NO: 3290gaaacaacgagaacattat 9432 9451 SEQ ID NO: 4630 ataaactgcaagatttttc 1360813627 1 4 SEQ ID NO: 3291 ttcttgaaaacgacaaagc 9599 9618 SEQ ID NO: 4631gctttccaatgaccaagaa 11065 11084 1 4 SEQ ID NO: 3292 ataagaaaaacaaacacag9648 9667 SEQ ID NO: 4632 ctgtgctttgtgagtttat 9690 9709 1 4 SEQ ID NO:3293 aaaacaaacacaggcattc 9654 9673 SEQ ID NO: 4633 gaatttgaaagttcgtttt9280 9299 1 4 SEQ ID NO: 3294 gcattccatcacaaatcct 9667 9686 SEQ ID NO:4634 aggaagtggccctgaatgc 10971 10990 1 4 SEQ ID NO: 3295tttgaaaaaaacagaaaca 9740 9759 SEQ ID NO: 4635 tgttgaaagatttatcaaa 1293312952 1 4 SEQ ID NO: 3296 caatgcattagattttgtc 9757 9776 SEQ ID NO: 4636gacaagaaaaaggggattg 10279 10298 1 4 SEQ ID NO: 3297 caaagctgaaaaatctcag9817 9836 SEQ ID NO: 4637 ctgagaacttcatcatttg 11438 11457 1 4 SEQ ID NO:3298 cctggatacactgttccag 9863 9882 SEQ ID NO: 4638 ctggacttctctagtcagg8810 8829 1 4 SEQ ID NO: 3299 gttgaagtgtctccattca 9890 9909 SEQ ID NO:4639 tgaatctggctccctcaac 9046 9065 1 4 SEQ ID NO: 3300tttctccatcctaggttct 9964 9983 SEQ ID NO: 4640 agaatccagatacaagaaa 68936912 1 4 SEQ ID NO: 3301 ttctccatcctaggttctg 9965 9984 SEQ ID NO: 4641cagaatccagatacaagaa 6892 6911 1 4 SEQ ID NO: 3302 tcattagagctgccagtcc10019 10038 SEQ ID NO: 4642 ggacagtgaaatattatga 13305 13324 1 4 SEQ IDNO: 3303 tgctgaactttttaaccag 10177 10196 SEQ ID NO: 4643ctggatgtaaccaccagca 11186 11205 1 4 SEQ ID NO: 3304 ctcctttcttcatcttcat10214 10233 SEQ ID NO: 4644 atgaagcttgctccaggag 13772 13791 1 4 SEQ IDNO: 3305 tgtcattgatgcactgcag 10234 10253 SEQ ID NO: 4645ctgcgctaccagaaagaca 12080 12099 1 4 SEQ ID NO: 3306 tgatgcactgcagtacaaa10240 10259 SEQ ID NO: 4646 tttgagttgcccaccatca 11666 11685 1 4 SEQ IDNO: 3307 agctctgtctctgagcaac 10309 10328 SEQ ID NO: 4647gttgaccacaagcttagct 10547 10566 1 4 SEQ ID NO: 3308 agccgaaattccaattttg10408 10427 SEQ ID NO: 4648 caaagctggcaccagggct 13971 13990 1 4 SEQ IDNO: 3309 ttgagaatgaatttcaagc 10424 10443 SEQ ID NO: 4649gcttcaggaagcttctcaa 13216 13235 1 4 SEQ ID NO: 3310 aaacctactgtctcttcct10469 10488 SEQ ID NO: 4650 aggaaggccaagccagttt 12591 12610 1 4 SEQ IDNO: 3311 tacttttccattgagtcat 10583 10602 SEQ ID NO: 4651atgattatgtcaacaagta 12363 12382 1 4 SEQ ID NO: 3312 tcaggtccatgcaagtcag10918 10937 SEQ ID NO: 4652 ctgacatcttaggcactga 5001 5020 1 4 SEQ ID NO:3313 atgcaagtcagcccagttc 10926 10945 SEQ ID NO: 4653 gaactcagaaggatggcat14002 14021 1 4 SEQ ID NO: 3314 tgaatgctaacactaagaa 10983 11002 SEQ IDNO: 4654 ttctcaattttgattttca 8526 8545 1 4 SEQ ID NO: 3315agaagatcagatggaaaaa 11004 11023 SEQ ID NO: 4655 ttttctaaatggaacttct12173 12192 1 4 SEQ ID NO: 3316 ggctattcattctccatcc 11264 11283 SEQ IDNO: 4656 ggatctaaatgcagtagcc 11632 11651 1 4 SEQ ID NO: 3317aaagttttggctgataaat 11288 11307 SEQ ID NO: 4657 atttcttaaacattccttt 94899508 1 4 SEQ ID NO: 3318 agttttggctgataaattc 11290 11309 SEQ ID NO: 4658gaatctggctccctcaact 9047 9066 1 4 SEQ ID NO: 3319 ctgggctgaaactaaatga11316 11335 SEQ ID NO: 4659 tcattctgggtctttccag 11035 11054 1 4 SEQ IDNO: 3320 cagagaaatacaaatctat 11413 11432 SEQ ID NO: 4660atagcatggacttcttctg 8873 8892 1 4 SEQ ID NO: 3321 gaggtaaaattccctgaag11480 11499 SEQ ID NO: 4662 cttctggcttgctaacctc 12306 12325 1 4 SEQ IDNO: 3322 cttttttgagataaccgtg 11545 11564 SEQ ID NO: 4663cacggagttactgaaaaag 13723 13742 1 4 SEQ ID NO: 3323 gctggaattgtcattcctt11735 11754 SEQ ID NO: 4664 aaggcatctccacctcagc 12102 12121 1 4 SEQ IDNO: 3324 gtgtataatgccacttgga 11795 11814 SEQ ID NO: 4665tccaagatgagatcaacac 13104 13123 1 4 SEQ ID NO: 3325 attccacatgcagctcaac11859 11878 SEQ ID NO: 4668 gttgagaagccccaagaat 6254 6273 1 4 SEQ ID NO:3326 tgaagaagatggcaaattt 11992 12011 SEQ ID NO: 4667 aaattctcttttcttttca9220 9239 1 4 SEQ ID NO: 3327 atcaaaagcccagcgttca 12050 12069 SEQ ID NO:4668 tgaaagtcaagcatctgat 12669 12688 1 4 SEQ ID NO: 3328gtgggcatggatatggatg 12143 12162 SEQ ID NO: 4669 catccttaacaccttccac 80718090 1 4 SEQ ID NO: 3329 aaatggaacttctactaca 12179 12198 SEQ ID NO: 4670tgtaccataagccatattt 10088 10107 1 4 SEQ ID NO: 3330 aaaaactcaccatattcaa12219 12238 SEQ ID NO: 4671 ttgatgttagagtgctttt 6993 7012 1 4 SEQ ID NO:3331 ctgagaagaaatctgcaga 12428 12447 SEQ ID NO: 4672 tctgcacagaaatattcag13447 13466 1 4 SEQ ID NO: 3332 acaatgctgagtgggttta 12447 12466 SEQ IDNO: 4673 taaatggagtctttattgt 14086 14105 1 4 SEQ ID NO: 3333caatgctgagtgggtttat 12448 12467 SEQ ID NO: 4674 ataaatggagtctttattg14085 14104 1 4 SEQ ID NO: 3334 ttaggcaaattgatgatat 12477 12496 SEQ IDNO: 4675 atattgtcagtgcctctaa 13392 13411 1 4 SEQ ID NO: 3335ataaactaatagatgtaat 12897 12916 SEQ ID NO: 4676 attactatgaaaaatttat13641 13660 1 4 SEQ ID NO: 3336 ccaactaatagaagataac 13039 13058 SEQ IDNO: 4677 gttattttgctaaacttgg 14052 14071 1 4 SEQ ID NO: 3337ttaattatatccaagatga 13095 13114 SEQ ID NO: 4678 tcatcctctaattttttaa13800 13819 1 4 SEQ ID NO: 3338 tttaaattgttgaaagaaa 13151 13170 SEQ IDNO: 4679 tttcatttgaaagaataaa 7032 7051 1 4 SEQ ID NO: 3339aagttcaatgaatttattc 13190 13209 SEQ ID NO: 4680 gaataccaatgctgaactt10168 10187 1 4 SEQ ID NO: 3340 ttgaagaaaagatagtcag 13326 13345 SEQ IDNO: 4681 ctgagagaagtgtcttcaa 12407 12426 1 4 SEQ ID NO: 3341acttccattctgaatatat 13377 13396 SEQ ID NO: 4682 atatctggaaccttgaagt10737 10756 1 4 SEQ ID NO: 3342 cacagaaatattcaggaat 13451 13470 SEQ IDNO: 4683 attccctgaagttgatgtg 11488 11507 1 4 SEQ ID NO: 3343ccattgcgacgaagaaaat 13560 13579 SEQ ID NO: 4684 atttttattcctgccatgg10103 10122 1 4 SEQ ID NO: 3344 tataaactgcaagattttt 13607 13626 SEQ IDNO: 4685 aaaattcaaactgcctata 13873 13892 1 4 SEQ ID NO: 3345tctgattactatgaaaaat 13637 13656 SEQ ID NO: 4686 atttgtaagaaaatacaga 64366455 1 4 SEQ ID NO: 3346 ggagttactgaaaaagctg 13726 13745 SEQ ID NO: 4687cagcatgcctagtttctcc 9952 9971 1 4 SEQ ID NO: 3347 tgaagcttgctccaggaga13773 13792 SEQ ID NO: 4688 tctcctttcttcatcttca 10213 10232 1 4 SEQ IDNO: 3348 tgaactggacctgcaccaa 13955 13974 SEQ ID NO: 4689ttggtagagcaagggttca 7856 7875 1 4 SEQ ID NO: 3349 ttgctaaacttgggggagg14058 14077 SEQ ID NO: 4690 cctcctacagtggtggcaa 4230 4249 1 4 SEQ ID NO:3350 gattcgaatatcaaattca 4412 4431 SEQ ID NO: 4691 tgaaaacgacaaagcaatc9603 9622 3 3 SEQ ID NO: 3351 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO:4692 acttttctaaacttgaaat 9063 9082 3 3 SEQ ID NO: 3352tctcggttgctgccgctga 33 52 SEQ ID NO: 4693 tcagcccagccatttgaga 9236 92552 3 SEQ ID NO: 3353 gctgaggagcccgcccagc 47 66 SEQ ID NO: 4694gctggatgtaaccaccagc 11185 11204 2 3 SEQ ID NO: 3354 ctggtctgtccaaaagatg227 246 SEQ ID NO: 4695 catcagaaccattgaccag 8134 8153 2 3 SEQ ID NO:3355 ctgagagttccagtggagt 291 310 SEQ ID NO: 4696 actcaatggtgaaattcag7465 7484 2 3 SEQ ID NO: 3356 cagtgcaccctgaaagagg 404 423 SEQ ID NO:4697 cctcacttcctttggactg 8977 8996 2 3 SEQ ID NO: 3357ctctgaggagtttgctgca 472 491 SEQ ID NO: 4698 tgcaaacttgacttcagag 1139911418 2 3 SEQ ID NO: 3358 acatcaagaggggcatcat 582 601 SEQ ID NO: 4699atgacgttcttgagcatgt 7050 7069 2 3 SEQ ID NO: 3359 ctgatcagcagcagccagt830 849 SEQ ID NO: 4700 actggacttctctagtcag 8809 8828 2 3 SEQ ID NO:3360 ggacgctaagaggaagcat 865 884 SEQ ID NO: 4701 atgcctacgttccatgtcc11354 11373 2 3 SEQ ID NO: 3361 agctgttttgaagactctc 1087 1106 SEQ ID NO:4702 gagaagtgtcttcaaagct 12411 12430 2 3 SEQ ID NO: 3362tgaaaaaactaaccatctc 1113 1132 SEQ ID NO: 4703 gagatcaacacaatcttca 1311213131 2 3 SEQ ID NO: 3363 ctgagctgagaggcctcag 1176 1195 SEQ ID NO: 4704ctgaattactgcacctcag 3035 3054 2 3 SEQ ID NO: 3364 tgaaacgtgtgcatgccaa1311 1330 SEQ ID NO: 4705 ttggtagagcaagggttca 7856 7875 2 3 SEQ ID NO:3365 ccttgtatgcgctgagcca 1440 1459 SEQ ID NO: 4706 tggcactgtttggagaagg9138 9157 2 3 SEQ ID NO: 3366 aggagctgctggacattgc 1500 1519 SEQ ID NO:4707 gcaagtcagcccagttcct 10928 10947 2 3 SEQ ID NO: 3367atttgattctgcgggtcat 1575 1594 SEQ ID NO: 4708 atgaaaccaatgacaaaat 74287447 2 3 SEQ ID NO: 3368 tccagaactcaagtcttca 1627 1646 SEQ ID NO: 4709tgaaatacaatgctctgga 5520 8539 2 3 SEQ ID NO: 3369 ggttcttcttcagactttc1744 1763 SEQ ID NO: 4710 gaaataccaagtcaaaacc 10455 10474 2 3 SEQ ID NO:3370 gttgatgaggagtccttca 1810 1829 SEQ ID NO: 4711 tgaaaaagctgcaatcaac13734 13753 2 3 SEQ ID NO: 3371 tccaagatctgaaaaagtt 1941 1960 SEQ ID NO:4712 aactgcttctccaaatgga 3552 3571 2 3 SEQ ID NO: 3372agttagtgaaagaagttct 1958 1975 SEQ ID NO: 4713 agaattcataatcccaact 82758294 2 3 SEQ ID NO: 3373 gaagggaatcttatatttg 2084 2103 SEQ ID NO: 4714caaaacctactgtctcttc 10487 10486 2 3 SEQ ID NO: 3374 ggaagctctttttgggaag2221 2240 SEQ ID NO: 4715 cttcacataccagaattcc 8324 8343 2 3 SEQ ID NO:3375 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4716 aacaaacacaggcattcca9656 9675 2 3 SEQ ID NO: 3376 gatttgaaatccaaagaag 2408 2427 SEQ ID NO:4717 cttcatgtccctagaaatc 10037 10056 2 3 SEQ ID NO: 3377tccaaagaagtcccggaag 2417 2438 SEQ ID NO: 4718 cttcagcctgctttctgga 49514970 2 3 SEQ ID NO: 3378 aggaagggctcaaagaatg 2570 2569 SEQ ID NO: 4719cattagagctgccagtcct 10020 10039 2 3 SEQ ID NO: 3379 agaatgacttttttcttca2583 2602 SEQ ID NO: 4720 tgaagatgacgacttttct 12160 12179 2 3 SEQ ID NO:3380 tttgtgacaaatatgggca 2765 2784 SEQ ID NO: 4721 tgccagtttgaaaaacaaa11815 11834 2 3 SEQ ID NO: 3381 ctgaggctaccatgacatt 3252 3271 SEQ ID NO:4722 aatgtcagctcttgttcag 10903 10922 2 3 SEQ ID NO: 3382gtagataccaaaaaaatga 3668 3687 SEQ ID NO: 4723 tcatttgccctcaacctac 1146011469 2 3 SEQ ID NO: 3383 aaatgacttccaatttccc 3681 3700 SEQ ID NO: 4724gggaactgttgaaagattt 12927 12946 2 3 SEQ ID NO: 3384 atgacttccaatttccctg3683 3702 SEQ ID NO: 4725 caggagaacttactatcat 13785 13804 2 3 SEQ ID NO:3385 atctgccatctcgagagtt 4104 4123 SEQ ID NO: 4726 aactcctccactgaaagat9547 9566 2 3 SEQ ID NO: 3386 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO:4727 acttccgtttaccagaaat 8247 8266 2 3 SEQ ID NO: 3387gcagagcttggcctctctg 5135 5154 SEQ ID NO: 4728 cagagctttctgccactgc 1351813537 2 3 SEQ ID NO: 3388 atatgctgaaatgaaattt 5353 5372 SEQ ID NO: 4729aaattcaaactgcctatat 13874 1389 2 3 SEQ ID NO: 3389 tcaaaacttgacaacattt5420 5439 SEQ ID NO: 4730 aaatacttccacaaattga 8780 8799 2 3 SEQ ID NO:3390 cagtgacctgaaatacaat 5512 5531 SEQ ID NO: 4731 attgaacatccccaaactg8794 8813 2 3 SEQ ID NO: 3391 tacaaatggcaatgggaaa 5848 5867 SEQ ID NO:4732 tttcaactgcctttgtgta 11229 11248 2 3 SEQ ID NO: 3392cttttgtaaagtatgataa 6285 6304 SEQ ID NO: 4733 ttattgctgaatccaaaag 1365613675 2 3 SEQ ID NO: 3393 ttgtaaagtatgataaaaa 6288 6307 SEQ ID NO: 4734ttttcaagcaaatgcacaa 8539 8556 2 3 SEQ ID NO: 3394 tccattaacctcccatttt6320 6339 SEQ ID NO: 4735 aaaagaaaattttgctgga 10755 10775 2 3 SEQ ID NO:3395 gattatctgaattcattca 6488 6507 SEQ ID NO: 4736 tgaagtagaccaacaaatc7162 7181 2 3 SEQ ID NO: 3396 aattgggagagacaagttt 6606 6525 SEQ ID NO:4737 aaactaaatgatctaaatt 11324 11343 2 3 SEQ ID NO: 3397atttgaaaatagctattgc 6698 6715 SEQ ID NO: 4738 gcaatttctgcacagaaat 1344113460 2 3 SEQ ID NO: 3398 tgagcatgtcaaacacttt 7060 7079 SEQ ID NO: 4739aaagccattcagtctctca 12971 12990 2 3 SEQ ID NO: 3399 ttgaagatgttaacaaatt7356 7375 SEQ ID NO: 4740 aattccatatgaaagtcaa 12660 12679 2 3 SEQ ID NO:3400 acttgtcacctacatttct 7783 7772 SEQ ID NO: 4741 agaatattttgatccaagt13278 13295 2 3 SEQ ID NO: 3401 gttttccacaccagaattt 8050 8089 SEQ ID NO:4742 aaatctggatttcttaaac 9481 9500 2 3 SEQ ID NO: 3402ataagtacaaccaaaattt 9405 9424 SEQ ID NO: 4743 aaataaatggagtctttat 1408314102 2 3 SEQ ID NO: 3403 cgggacctgcggggctgag 8 27 SEQ ID NO: 4744ctcagttaactgtgtcccg 11571 11590 1 3 SEQ ID NO: 3404 agtgcccttctcggttgct28 44 SEQ ID NO: 4745 agcatctgattgactcact 12878 12697 1 3 SEQ ID NO:3405 gctgaggagcccgcccagc 47 66 SEQ ID NO: 4746 gctgattgaggtgtccagc 12251244 1 3 SEQ ID NO: 3406 gaggagcccgcccagccag 50 69 SEQ ID NO: 4747ctggatcacagagtccctc 3752 3771 1 3 SEQ ID NO: 3407 gggccgcgaggccgaggcc 7291 SEQ ID NO: 4748 ggccctgatccccgagccc 1383 1382 1 3 SEQ ID NO: 3408ccaggccgcagcccaggag 89 108 SEQ ID NO: 4749 ctcccggagccaaggctgg 2682 27011 3 SEQ ID NO: 3409 ggagccgccccaccgcagc 104 123 SEQ ID NO: 4750gctgttttgaagactctcc 1085 1107 1 3 SEQ ID NO: 3410 gaagaggaaatgctggaaa200 219 SEQ ID NO: 4751 tttcaagttcctgaccttc 8309 8328 1 3 SEQ ID NO:3411 caaaagatgcgacccgatt 237 256 SEQ ID NO: 4752 aatcttattggggattttg7085 7104 1 3 SEQ ID NO: 3412 attcaagcacctccggaag 253 272 SEQ ID NO:4753 cttccacatttcaaggaat 10067 10086 1 3 SEQ ID NO: 3413gttccagtggagtccctgg 297 316 SEQ ID NO: 4754 ccagcaagtacctgagaac 86108629 1 3 SEQ ID NO: 3414 gactgctgattcaagaagt 316 335 SEQ ID NO: 4755acttgaagaaaagatagtc 13324 13343 1 3 SEQ ID NO: 3415 gtgccaccaggatcaactg333 352 SEQ ID NO: 4756 cagtgaagctgcagggcac 10704 10723 1 3 SEQ ID NO:3416 gatcaactgcaaggttgag 343 362 SEQ ID NO: 4757 ctcacctccacctctgatc4748 4767 1 3 SEQ ID NO: 3417 actgcaaggttgagctgga 348 367 SEQ ID NO:4758 tccactcacatcctccagt 1289 1308 1 3 SEQ ID NO: 3418ccagctctgcagcttcatc 373 392 SEQ ID NO: 4759 gatgtggtcacctacctgg 13431382 1 3 SEQ ID NO: 3419 agcttcatcctgaagacca 383 402 SEQ ID NO: 4760tggtgctggagaatgagct 5112 5131 1 3 SEQ ID NO: 3420 cttcatcctgaagaccagc385 404 SEQ ID NO: 4761 gctggagtaaaactggaag 2696 2715 1 3 SEQ ID NO:3421 ccagccagtgcaccctgaa 399 418 SEQ ID NO: 4762 ttcaagatgactgcactgg1539 1558 1 3 SEQ ID NO: 3422 cagtgcaccctgaaagagg 404 423 SEQ ID NO:4763 cctcacagagctatcactg 5230 5249 1 3 SEQ ID NO: 3423tggcttcaaccctgagggc 427 448 SEQ ID NO: 4764 gcccactggtcgcctgcca 36333552 1 3 SEQ ID NO: 3424 cttcaaccctgagggcaaa 430 449 SEQ ID NO: 4765tttgagccaacattggaag 2207 2226 1 3 SEQ ID NO: 3425 ttcaaccctgagggcaaag431 450 SEQ ID NO: 4766 ctttgacaggcattttgaa 9727 9746 1 3 SEQ ID NO:3426 cttgctgaagaaaaccaag 451 470 SEQ ID NO: 4767 cttgaaattcaatcacaag9074 9093 1 3 SEQ ID NO: 3427 tgctgaagaaaaccaagaa 453 472 SEQ ID NO:4768 ttctgctgccttatcagca 5647 5666 1 3 SEQ ID NO: 3428ttgctgcagccatgtccag 483 502 SEQ ID NO: 4769 ctggtcagtttgcaagcaa 30043023 1 3 SEQ ID NO: 3429 tgctgcagccatgtccagg 484 503 SEQ ID NO: 4770cctggtcagtttgcaagca 3003 3022 1 3 SEQ ID NO: 3430 agccatgtccaggtatgag490 509 SEQ ID NO: 4771 ctcacatcctccagtggct 1293 1312 1 3 SEQ ID NO:3431 agctcaagctggccattcc 507 528 SEQ ID NO: 4772 ggaactaccacaaaaagct7489 7508 1 3 SEQ ID NO: 3432 agaagggaagcaggttttc 526 545 SEQ ID NO:4773 gaaatcttcaatttattct 13821 13840 1 3 SEQ ID NO: 3433aagggaagcaggttttcct 528 547 SEQ ID NO: 4774 aggacaccaaaataacctt 75727591 1 3 SEQ ID NO: 3434 agaaagatgaacctactta 555 574 SEQ ID NO: 4775taagaactttgccacttct 4852 4871 1 3 SEQ ID NO: 3435 atcctgaacatcaagaggg575 594 SEQ ID NO: 4776 ccctaacagatttgaggat 7977 7996 1 3 SEQ ID NO:3436 tcctgaacatcaagagggg 576 595 SEQ ID NO: 4777 cccctaacagatttgagga7976 7995 1 3 SEQ ID NO: 3437 ctgaacatcaagaggggca 578 597 SEQ ID NO:4778 tgcctgcctttgaagtcag 7908 7927 1 3 SEQ ID NO: 3438aacatcaagaggggcatca 551 600 SEQ ID NO: 4779 tgataaaaaccaagatgtt 62986317 1 3 SEQ ID NO: 3439 acatcaagaggggcatcat 582 601 SEQ ID NO: 4780atgataaaaaccaagatgt 6297 6316 1 3 SEQ ID NO: 3440 tcatttctgccctcctggt597 616 SEQ ID NO: 4781 accaccagtttgtagatga 7413 7432 1 3 SEQ ID NO:3441 ttcccccagagacagaaga 615 634 SEQ ID NO: 4782 tcttccacatttcaaggaa10066 10055 1 3 SEQ ID NO: 3442 gaagaagccaagcaagtgt 629 648 SEQ ID NO:4783 acaccttccacattccttc 8079 8098 1 3 SEQ ID NO: 3443ttgtttctggataccgtgt 647 666 SEQ ID NO: 4784 acactaaatacttccacaa 87758794 1 3 SEQ ID NO: 3444 tgtatggaaactgctccac 663 682 SEQ ID NO: 4785gtggaggcaacacattaca 2928 2947 1 3 SEQ ID NO: 3445 aaactgctccactcacttt670 689 SEQ ID NO: 4786 aaagaaacagcatttgttt 4540 4559 1 3 SEQ ID NO:3446 actcactttaccgtcaaga 680 699 SEQ ID NO: 4787 tcttacttttccattgagt10580 10599 1 3 SEQ ID NO: 3447 ctttaccgtcaagacgagg 685 704 SEQ ID NO:4788 cctccagctcctgggaaag 2491 2510 1 3 SEQ ID NO: 3448ttaccgtcaagacgaggaa 687 706 SEQ ID NO: 4789 ttcctaaagctggatgtaa 1117711196 1 3 SEQ ID NO: 3449 acgaggaagggcaatgtgg 698 717 SEQ ID NO: 4790ccacaagtcatcatctcgt 5964 5983 1 3 SEQ ID NO: 3450 cgaggaagggcaatgtggc699 718 SEQ ID NO: 4791 gccagaagtgagatcctcg 3515 3534 1 3 SEQ ID NO:3451 gaggaagggcaatgtggca 700 719 SEQ ID NO: 4792 tgccagtctccatgacctc2476 2495 1 3 SEQ ID NO: 3452 ggaagggcaatgtggcaac 702 721 SEQ ID NO:4793 gttgctcttaaggacttcc 13364 13383 1 3 SEQ ID NO: 3453gaagggcaatgtggcaaca 703 722 SEQ ID NO: 4794 tgttgatgaggagtccttc 18091828 1 3 SEQ ID NO: 3454 caggcatcagcccacttgc 777 796 SEQ ID NO: 4795gcaagtctttcctggcctg 3019 3038 1 3 SEQ ID NO: 3455 aggcatcagcccacttgct778 797 SEQ ID NO: 4796 agcaagtctttcctggcct 3018 3037 1 3 SEQ ID NO:3456 tcagcccacttgctctcat 783 802 SEQ ID NO: 4797 atgaaagtcaagcatctga12668 12687 1 3 SEQ ID NO: 3457 gtcaactctgatcagcagc 823 842 SEQ ID NO:4798 gctgactttaaaatctgac 4819 4838 1 3 SEQ ID NO: 3458ggacgctaagaggaagcat 865 884 SEQ ID NO: 4799 atgcactgtttctgagtcc 93399358 1 3 SEQ ID NO: 3459 aaggagcaacacctcttcc 802 921 SEQ ID NO: 4800ggaatatcttagcatcctt 13465 13484 1 3 SEQ ID NO: 3460 aggagcaacacctcttcct903 922 SEQ ID NO: 4801 aggaatatcttagcatcct 13464 13483 1 3 SEQ ID NO:3461 caacacctcttcctgcctt 908 927 SEQ ID NO: 4802 aaggctgactctgtggttg4292 4311 1 3 SEQ ID NO: 3462 aacacctcttcctgccttt 909 928 SEQ ID NO:4803 aaagcaggccgaagctgtt 1076 1094 1 3 SEQ ID NO: 3463acaagaataagtatgggat 933 952 SEQ ID NO: 4804 atccatgatctacatttgt 67946813 1 3 SEQ ID NO: 3464 caagaataagtatgggatg 934 953 SEQ ID NO: 4805catcactttacaagccttg 1246 1265 1 3 SEQ ID NO: 3465 tagcacaagtgacacagac954 973 SEQ ID NO: 4806 gtctcttcgttctatgcta 4592 4611 1 3 SEQ ID NO:3466 agcacaagtgacacagact 955 974 SEQ ID NO: 4807 agtctcttcgttctatgct4591 4610 1 3 SEQ ID NO: 3467 gcacaagtgacacagactt 956 975 SEQ ID NO:4808 aagtgtagtctcctggtgc 5099 5116 1 3 SEQ ID NO: 3468aacttgaagacacaccaaa 978 997 SEQ ID NO: 4809 tttgaggattccatcagtt 79878006 1 3 SEQ ID NO: 3469 gcttctttggtgaaggtac 1008 1027 SEQ ID NO: 4810gtacctacttttggcaagc 8372 8391 1 3 SEQ ID NO: 3470 ctttggtgaaggtactaag1012 1031 SEQ ID NO: 4811 cttatgggatttcctaaag 11167 11186 1 3 SEQ ID NO:3471 tactaagaagatgggcctc 1024 1043 SEQ ID NO: 4812 gagggtagtcataacagta10337 10356 1 3 SEQ ID NO: 3472 tttgagagcaccaaatcca 1046 1065 SEQ ID NO:4813 tggaagtgtcagtggcaaa 10380 10399 1 3 SEQ ID NO: 3473agagcaccaaatccacatc 1050 1069 SEQ ID NO: 4814 gatggatatgaccttctct 48764895 1 3 SEQ ID NO: 3474 agctgttttgaagactctc 1087 1106 SEQ ID NO: 4815gagaacatactgggcagct 5880 5899 1 3 SEQ ID NO: 3475 tgaaaaaactaaccatctc1113 1132 SEQ ID NO: 4816 gagaaaatcaatgccttca 7112 7131 1 3 SEQ ID NO:3476 gaaaaaactaaccatctct 1114 1133 SEQ ID NO: 4817 agagccaggtcgagctttc11052 11071 1 3 SEQ ID NO: 3477 tctgagcaaaatatccaga 1130 1149 SEQ ID NO:4818 tctgatgaggaaactcaga 12260 12279 1 3 SEQ ID NO: 3478tctcttcaataagctggtt 1156 1175 SEQ ID NO: 4819 aacctcccattttttgaga 63266345 1 3 SEQ ID NO: 3479 ctgagctgagaggcctcag 1176 1195 SEQ ID NO: 4820ctgatccccgagccctcag 1367 1386 1 3 SEQ ID NO: 3480 tgaagcagtcacatctctc1198 1217 SEQ ID NO: 4821 gagaaaatcaatgccttca 7112 7131 1 3 SEQ ID NO:3481 aagcagtcacatctctctt 1200 1219 SEQ ID NO: 4822 aagaggcagcttctggctt12297 12316 1 3 SEQ ID NO: 3482 ctctcttgccacagctgat 1212 1231 SEQ ID NO:4823 atcaaaagaagcccaagag 12946 12965 1 3 SEQ ID NO: 3483tcttgccacagctgattga 1215 1234 SEQ ID NO: 4824 tcaaagttaattgggaaga 1227912298 1 3 SEQ ID NO: 3484 cttgccacagctgattgag 1216 1235 SEQ ID NO: 4825ctcaattttgattttcaag 8528 8547 1 3 SEQ ID NO: 3485 tgaggtgtccagccccatc1231 1250 SEQ ID NO: 4826 gatggaaccctctccctca 4733 4752 1 3 SEQ ID NO:3486 tcagtgtggacagcctcag 1267 1286 SEQ ID NO: 4827 ctgacatcttaggcactga5001 5020 1 3 SEQ ID NO: 3487 acatcctccagtggctgaa 1296 1315 SEQ ID NO:4828 ttcagaagctaagcaatgt 7239 7258 1 3 SEQ ID NO: 3488gcacagcagctgcgagaga 1385 1404 SEQ ID NO: 4829 tctctgaaagacaacgtgc 1232312342 1 3 SEQ ID NO: 3489 cagcagctgcgagagatct 1388 1407 SEQ ID NO: 4830agataacattaaacagctg 13051 13070 1 3 SEQ ID NO: 3490 gcgagggatcagcgcagcc1415 1434 SEQ ID NO: 4831 ggctcaacacagacatcgc 5718 5737 1 3 SEQ ID NO:3491 aagacaaaccctacaggga 1478 1497 SEQ ID NO: 4832 tcccagaaaacctcttctt3938 3955 1 3 SEQ ID NO: 3492 caggagctgctggacattg 1499 1518 SEQ ID NO:4833 caatggagagtccaacctg 4660 4679 1 3 SEQ ID NO: 3493aggagctgctggacattgc 1500 1519 SEQ ID NO: 4834 gcaagggttcactgttcct 78647883 1 3 SEQ ID NO: 3494 ctgctggacattgctaatt 1505 1524 SEQ ID NO: 4835aattgggaagaagaggcag 12287 12306 1 3 SEQ ID NO: 3495 gattacacctatttgattc1565 1584 SEQ ID NO: 4836 gaatattttgagaggaatc 6353 6372 1 3 SEQ ID NO:3496 atttgattctgcgggtcat 1575 1594 SEQ ID NO: 4837 atgaagtagaccaacaaat7161 7180 1 3 SEQ ID NO: 3497 tctgcgggtcattggaaat 1582 1601 SEQ ID NO:4838 atttgtaagaaaatacaga 6436 6455 1 3 SEQ ID NO: 3498aaccatggagcagttaact 1609 1628 SEQ ID NO: 4839 agtttctccatcctaggtt 99629981 1 3 SEQ ID NO: 3499 ggagcagttaactccagaa 1615 1634 SEQ ID NO: 4840ttctgaaaatccaatctcc 8400 8419 1 3 SEQ ID NO: 3500 actccagaactcaagtctt1625 1644 SEQ ID NO: 4841 aagatcgcagactttgagt 11654 11673 1 3 SEQ ID NO:3501 tccagaactcaagtcttca 1627 1646 SEQ ID NO: 4842 tgaactcagaagaattgga1920 1939 1 3 SEQ ID NO: 3502 aagtacaaagccatcactg 1663 1682 SEQ ID NO:4843 cagtcatgtagaaaaactt 4429 4448 1 3 SEQ ID NO: 3503gccatcactgatgatccag 1672 1691 SEQ ID NO: 4844 ctggaactctctccatggc 1088310902 1 3 SEQ ID NO: 3504 ccatcactgatgatccaga 1673 1692 SEQ ID NO: 4845tctgaactcagaaggatgg 13999 14018 1 3 SEQ ID NO: 3505 atccagaaagctgccatcc1665 1704 SEQ ID NO: 4846 ggatttcctaaagctggat 11173 11192 1 3 SEQ ID NO:3506 cagaaagctgccatccagg 1668 1707 SEQ ID NO: 4847 cctgaaatacaatgctctg5518 5537 1 3 SEQ ID NO: 3507 acaaggaccaggaggttct 1731 1750 SEQ ID NO:4848 agaaacagcatttgtttgt 4542 4651 1 3 SEQ ID NO: 3508aggaccaggaggttcttct 1734 1753 SEQ ID NO: 4849 agaagctaagcaatgtcct 72427261 1 3 SEQ ID NO: 3509 accaggaggttcttcttca 1737 1756 SEQ ID NO: 4850tgaaggctgactctgtggt 4290 4309 1 3 SEQ ID NO: 3510 tcttcagactttccttgat1750 1769 SEQ ID NO: 4851 atcaggaagggctcaaaga 2567 2586 1 3 SEQ ID NO:3511 ttcagactttccttgatga 1752 1771 SEQ ID NO: 4852 tcattactcctgggctgaa11307 11326 1 3 SEQ ID NO: 3512 gttgatgaggagtccttca 1810 1829 SEQ ID NO:4853 tgaatctggctccctcaac 9046 9065 1 3 SEQ ID NO: 3513cttcacaggcagatattaa 1824 1843 SEQ ID NO: 4854 ttaatcgagaggtatgaag 71487167 1 3 SEQ ID NO: 3514 ttcacaggcagatattaac 1825 1844 SEQ ID NO: 4855gttaatcgagaggtatgaa 7147 7166 1 3 SEQ ID NO: 3515 ggcagatattaacaaaatt1831 1850 SEQ ID NO: 4856 aattgcattagatgatgcc 6589 6808 1 3 SEQ ID NO:3516 atattaacaaaattgtcca 1836 1855 SEQ ID NO: 4857 tggagtttgtgacaaatat2760 2779 1 3 SEQ ID NO: 3517 acaaaattgtccaaattct 1842 1861 SEQ ID NO:4858 agaaacagcatttgtttgt 4542 4561 1 3 SEQ ID NO: 3518gagcaagtgaagaactttg 1877 1896 SEQ ID NO: 4859 caaatgacatgatgggctc 53345353 1 3 SEQ ID NO: 3519 gtgaagaactttgtggctt 1883 1902 SEQ ID NO: 4860aagcatctgattgactcac 12677 12698 1 3 SEQ ID NO: 3520 agaactttgtggcttccca1887 1906 SEQ ID NO: 4861 tgggcctgccccagattct 8909 8928 1 3 SEQ ID NO:3521 tttgtggcttcccatattg 1802 1911 SEQ ID NO: 4862 caataagatcaatagcaaa8998 9017 1 3 SEQ ID NO: 3522 tggcttcccatattgccaa 1896 1915 SEQ ID NO:4883 ttggctcacatgaaggcca 7631 7650 1 3 SEQ ID NO: 3523ttcccatattgccaatatc 1900 1919 SEQ ID NO: 4864 gatatacactagggaggaa 1274512764 1 3 SEQ ID NO: 3524 tcccatattgccaatatct 1901 1920 SEQ ID NO: 4865agatcaaagttaattggga 12276 12295 1 3 SEQ ID NO: 3525 ttgccaatatcttgaactc1908 1927 SEQ ID NO: 4866 gagtcccagtgcccagcaa 9352 9371 1 3 SEQ ID NO:3526 ttggatatccaagatctga 1934 1953 SEQ ID NO: 4857 tcagtataagtacaaccaa9400 9419 1 3 SEQ ID NO: 3527 tccaagatctgaaaaagtt 1941 1960 SEQ ID NO:4868 aacttccaactgtcatgga 1986 2005 1 3 SEQ ID NO: 3528ctgaaaaagttagtgaaag 1949 1968 SEQ ID NO: 4869 ctttgaagtcagtcttcag 79157934 1 3 SEQ ID NO: 3529 agttagtgaaagaagttct 1956 1975 SEQ ID NO: 4870agaatctcaacttccaact 1978 1997 1 3 SEQ ID NO: 3530 aatctcaacttccaactgt1980 1999 SEQ ID NO: 4871 acaggggtcctttatgatt 12350 12369 1 3 SEQ ID NO:3531 gtcatggacttcagaaaat 1997 2016 SEQ ID NO: 4872 atttgaaagaataaatgac7036 7055 1 3 SEQ ID NO: 3532 tcaactctacaaatctgtt 2029 2048 SEQ ID NO:4873 aacacattgaggctattga 6978 6997 1 3 SEQ ID NO: 3533aactctacaaatctgtttc 2031 2050 SEQ ID NO: 4874 gaaaaaggggattgaagtt 1028410303 1 3 SEQ ID NO: 3534 aaatagaagggaatcttat 2079 2098 SEQ ID NO: 4875ataagcaaactgttaattt 5457 5476 1 3 SEQ ID NO: 3535 agaagggaatcttatattt2083 2102 SEQ ID NO: 4876 aaatgcactgctgcgttct 4900 4919 1 3 SEQ ID NO:3536 gaagggaatcttatatttg 2084 2103 SEQ ID NO: 4877 caaaaacattttcaacttc5287 5306 1 3 SEQ ID NO: 3537 tgatccaaataactacctt 2101 2120 SEQ ID NO:4878 aaggaagaaagaaaaatca 3461 3480 1 3 SEQ ID NO: 3538tggatttgcttcagctgac 2158 2177 SEQ ID NO: 4879 gtcagcccagttccttcca 1093210951 1 3 SEQ ID NO: 3539 tttgcttcagctgacctca 2162 2181 SEQ ID NO: 4880tgaggaaactcagatcaaa 12265 12284 1 3 SEQ ID NO: 3540 cttggaaggaaaaggcttt2191 2210 SEQ ID NO: 4881 aaagcattggtagagcaag 7850 7869 1 3 SEQ ID NO:3541 tggaaggaaaaggctttga 2193 2212 SEQ ID NO: 4882 tcaagtctgtgggattcca4086 4105 1 3 SEQ ID NO: 3542 ggctttgagccaacattgg 2204 2223 SEQ ID NO:4883 ccaagaggtatttaaagcc 12958 12977 1 3 SEQ ID NO: 3543tgagccaacattggaagct 2209 2228 SEQ ID NO: 4884 agctttctgccactgctca 1352113540 1 3 SEQ ID NO: 3544 gagccaacattggaagctc 2210 2229 SEQ ID NO: 4885gagctttctgccactgctc 13520 13539 1 3 SEQ ID NO: 3545 aacattggaagctcttttt2215 2234 SEQ ID NO: 4886 aaaagaaacagcatttgtt 4539 4558 1 3 SEQ ID NO:3546 tggaagctctttttgggaa 2220 2239 SEQ ID NO: 4887 ttccggcacgtgggttcca3785 3804 1 3 SEQ ID NO: 3547 ctctttttgggaagcaagg 2226 2245 SEQ ID NO:4888 ccttactgactttgcagag 7799 7817 1 3 SEQ ID NO: 3548tttttgggaagcaaggatt 2229 2248 SEQ ID NO: 4889 aatcattgaaaaattaaaa 67306749 1 3 SEQ ID NO: 3549 ttttcccagacagtgtcaa 2247 2265 SEQ ID NO: 4890ttgatgaaatcattgaaaa 6723 6742 1 3 SEQ ID NO: 3550 ttggctataccaaagatga2331 2360 SEQ ID NO: 4891 tcattgctcccggagccaa 2678 2695 1 3 SEQ ID NO:3551 ataccaaagatgataaaca 2337 2358 SEQ ID NO: 4892 tgttgcttttgtaaagtat6280 6299 1 3 SEQ ID NO: 3552 gagcaggatatggtaaatg 2357 2376 SEQ ID NO:4893 catttcagccttcgggctc 4262 4281 1 3 SEQ ID NO: 3553atggtaaatggaataatgc 2366 2385 SEQ ID NO: 4894 gcatgcctagtttctccat 99549973 1 3 SEQ ID NO: 3554 tggtaaatggaataatgct 2367 2386 SEQ ID NO: 4895agcacagtacgaaaaacca 10809 10828 1 3 SEQ ID NO: 3555 taaatggaataatgctcag2370 2389 SEQ ID NO: 4896 ctgaaagagatgaaattta 13067 13086 1 3 SEQ ID NO:3556 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4897 aacagatttgaggattcca7981 8000 1 3 SEQ ID NO: 3557 tcagtgttgagaagctgat 2355 2404 SEQ ID NO:4898 atcacaactcctccactga 9542 9561 1 3 SEQ ID NO: 3558cagtgttgagaagctgatt 2386 2405 SEQ ID NO: 4899 aatcacaactcctccactg 95419560 1 3 SEQ ID NO: 3559 agtgttgagaagctgatta 2387 2406 SEQ ID NO: 4900taatcacaactcctccact 9540 9559 1 3 SEQ ID NO: 3560 gattaaagatttgaaatcc2401 2420 SEQ ID NO: 4901 ggatactaagtaccaaatc 5874 6893 1 3 SEQ ID NO:3561 gatttgaaatccaaagaag 2408 2427 SEQ ID NO: 4902 cttccgtttaccagaaatc8248 8267 1 3 SEQ ID NO: 3562 atttgaaatccaaagaagt 2409 2428 SEQ ID NO:4903 acttccgtttaccagaaat 8247 8268 1 3 SEQ ID NO: 3563atccaaagaagtcccggaa 2416 2435 SEQ ID NO: 4904 ttccaatttccctgtggat 36883707 1 3 SEQ ID NO: 3564 tccaaagaagtcccggaag 2417 2436 SEQ ID NO: 4905cttccaatttccctgtgga 3887 3706 1 3 SEQ ID NO: 3565 agagcctacctccgcatct2438 2457 SEQ ID NO: 4906 agattaatccgctggctct 8571 8590 1 3 SEQ ID NO:3566 gagcctacctccgcatctt 2439 2458 SEQ ID NO: 4907 aagattaatccgctggctc8570 8589 1 3 SEQ ID NO: 3567 cttgggagaggagcttggt 2455 2474 SEQ ID NO:4908 accactgggacctaccaag 12527 12546 1 3 SEQ ID NO: 3568ggagcttggttttgccagt 2464 2483 SEQ ID NO: 4909 actggtggcaaaaccctcc 27342753 1 3 SEQ ID NO: 3569 ttggttttgccagtctcca 2469 2488 SEQ ID NO: 4910tggagaagccacactccaa 10771 10790 1 3 SEQ ID NO: 3570 cagtctccatgacctccag2479 2498 SEQ ID NO: 4911 ctggtcgcctgccaaactg 3538 3557 1 3 SEQ ID NO:3571 ctccatgacctccagctcc 2483 2502 SEQ ID NO: 4912 ggagtcattgctcccggag2672 2691 1 3 SEQ ID NO: 3572 ctgggaaagctgcttctga 2501 2520 SEQ ID NO:4913 tcagaaagctaccttccag 7939 7958 1 3 SEQ ID NO: 3573gaggtcatcaggaagggct 2561 2580 SEQ ID NO: 4914 agccagaagtgagatcctc 35143533 1 3 SEQ ID NO: 3574 aagaatgacttttttcttc 2582 2601 SEQ ID NO: 4915gaaggcatctgggagtctt 3835 3854 1 3 SEQ ID NO: 3575 cttttttcttcactacatc2590 2609 SEQ ID NO: 4916 gatgcttacaacactaaag 6107 6126 1 3 SEQ ID NO:3576 catcttcatggagaatgcc 2605 2624 SEQ ID NO: 4917 ggcacttccaaaattgatg10718 10737 1 3 SEQ ID NO: 3577 cttcatggagaatgccttt 2608 2627 SEQ ID NO:4918 aaagttaattgggaagaag 12281 12300 1 3 SEQ ID NO: 3578aatgcctttgaactcccca 2618 2637 SEQ ID NO: 4919 tgggctggcttcagccatt 57375756 1 3 SEQ ID NO: 3579 gcctttgaactccccactg 2621 2640 SEQ ID NO: 4920cagtctgaacattgcaggc 5383 5402 1 3 SEQ ID NO: 3580 caaggctggagtaaaactg2692 2711 SEQ ID NO: 4921 cagtgcaacgaccaacttg 5080 5099 1 3 SEQ ID NO:3581 tggagtaaaactggaagta 2698 2717 SEQ ID NO: 4922 tactccaacgccagctcca3059 3078 1 3 SEQ ID NO: 3582 ggaagtagccaacatgcag 2710 2729 SEQ ID NO:4923 ctgccatctcgagagttcc 4106 4125 1 3 SEQ ID NO: 3583tttgtgacaaatatgggca 2765 2784 SEQ ID NO: 4924 tgcctttgtgtacaccaaa 1123611255 1 3 SEQ ID NO: 3584 tgtgacaaatatgggcatc 2767 2786 SEQ ID NO: 4925gatgggtctctacgccaca 4385 4404 1 3 SEQ ID NO: 3585 ggacttcgctaggagtggg2794 2813 SEQ ID NO: 4926 cccaaggccacaggggtcc 12341 12360 1 3 SEQ ID NO:3586 gtggggtccagatgaacac 2808 2827 SEQ ID NO: 4927 gtgttctagacctctccac4179 4198 1 3 SEQ ID NO: 3587 ttccacgagtcgggtctgg 2834 2853 SEQ ID NO:4928 ccagaatctgtaccaggaa 12562 12581 1 3 SEQ ID NO: 3588agtcgggtctggaggctca 2841 2860 SEQ ID NO: 4929 tgagaactacgagctgact 48074826 1 3 SEQ ID NO: 3589 tcgggtctggaggctcatg 2843 2862 SEQ ID NO: 4930catgaaggccaaattccga 7639 7658 1 3 SEQ ID NO: 3590 aaaagctgggaagctgaag2869 2883 SEQ ID NO: 4931 cttccagacacctgatttt 7951 7970 1 3 SEQ ID NO:3591 aagctgaagtttatcattc 2879 2898 SEQ ID NO: 4932 gaatttacaattgttgctt6269 6288 1 3 SEQ ID NO: 3592 gagaccagtcaagctgctc 2908 2927 SEQ ID NO:4933 gagcttcaggaagcttctc 13214 13233 1 3 SEQ ID NO: 3593gcaacacattacatttggt 2934 2953 SEQ ID NO: 4934 accagtcagatattgttgc 1019110210 1 3 SEQ ID NO: 3594 acattacatttggtctcta 2939 2958 SEQ ID NO: 4935tagaatatgaactaaatgt 11889 11908 1 3 SEQ ID NO: 3595 cattacatttggtctctac2940 2959 SEQ ID NO: 4936 gtagctgagaaaatcaatg 7108 7125 1 3 SEQ ID NO:3596 aaacggaggtgatcccacc 2964 2983 SEQ ID NO: 4937 ggtggataccctgaagttt3205 3224 1 3 SEQ ID NO: 3597 attgagaacaggcagtcct 2987 3006 SEQ ID NO:4938 aggaaaagcgcacctcaat 12031 12050 1 3 SEQ ID NO: 3598tgagaacaggcagtcctgg 2989 3008 SEQ ID NO: 4939 ccagcttccccacatctca 83418360 1 3 SEQ ID NO: 3599 ctgcacctcaggcgcttac 3043 3002 SEQ ID NO: 4940gtaagaaaatacagagcag 6440 6459 1 3 SEQ ID NO: 3600 tccacagactccgcctcct3074 3093 SEQ ID NO: 4941 aggacagagccttggtgga 3192 3211 1 3 SEQ ID NO:3601 ctgaccggggacaccagat 3101 3120 SEQ ID NO: 4942 atctgatgaggaaactcag12259 12278 1 3 SEQ ID NO: 3602 tagagctggaactgaggcc 3120 3139 SEQ ID NO:4943 ggcctctctggggcatcta 5144 5163 1 3 SEQ ID NO: 3603ctatgagctccagagagag 3175 3194 SEQ ID NO: 4944 ctctcacaaaaaagtatag 65496568 1 3 SEQ ID NO: 3604 cttggtggataccctgaag 3202 3221 SEQ ID NO: 4945cttcaggaagcttctcaag 13217 13236 1 3 SEQ ID NO: 3605 ttgtaactcaagcagaagg3222 3241 SEQ ID NO: 4946 ccttacacaataatcacaa 9530 9549 1 3 SEQ ID NO:3606 taactcaagcagaaggtgc 3225 3244 SEQ ID NO: 4947 gcacctagctggaaagtta6955 6974 1 3 SEQ ID NO: 3607 gcagaaggtgcgaagcaga 3233 3252 SEQ ID NO:4948 tctgtgggattccatctgc 4091 4110 1 3 SEQ ID NO: 3608cagaaggtgcgaagcagac 3234 3253 SEQ ID NO: 4949 gtctgtgggattccatctg 40904109 1 3 SEQ ID NO: 3609 gtatgaccttgtccagtga 3288 3307 SEQ ID NO: 4950tcaccaacggagaacatac 10851 10870 1 3 SEQ ID NO: 3610 tatgaccttgtccagtgaa3289 3308 SEQ ID NO: 4951 ttcaccaacggagaacata 10850 10869 1 3 SEQ ID NO:3611 gaagtccaaattccggatt 3305 3324 SEQ ID NO: 4952 aatctcaagctttctcttc10052 10071 1 3 SEQ ID NO: 3612 gagggcaaaacgtcttaca 3371 3390 SEQ ID NO:4953 tgtacaactggtccgcctc 4215 4234 1 3 SEQ ID NO: 3613agggcaaaacgtcttacag 3372 3391 SEQ ID NO: 4954 ctgttaggacaccagccct 40624081 1 3 SEQ ID NO: 3614 gactcaccctggacattca 3390 3409 SEQ ID NO: 4955tgaaattcaatcacaagtc 9076 9095 1 3 SEQ ID NO: 3615 ctggacattcagaacaaga3398 3417 SEQ ID NO: 4956 tcttttcttttcagcccag 9226 9245 1 3 SEQ ID NO:3616 tcatgggcgacctaagttg 3435 3454 SEQ ID NO: 4957 caactgcagacatatatga6635 6654 1 3 SEQ ID NO: 3617 tgggcgacctaagttgtga 3438 3457 SEQ ID NO:4958 tcactccattaacctccca 6316 6335 1 3 SEQ ID NO: 3618agttgtgacacaaaggaag 3449 3468 SEQ ID NO: 4959 cttcttttccaattgaact 1383813857 1 3 SEQ ID NO: 3619 tgacacaaaggaagaaaga 3454 3473 SEQ ID NO: 4960tcttcatcttcatctgtca 10220 10239 1 3 SEQ ID NO: 3620 gacacaaaggaagaaagaa3455 3474 SEQ ID NO: 4961 ttcttcatcttcatctgtc 10219 10238 1 3 SEQ ID NO:3621 ggaagaaagaaaaatcaag 3463 3482 SEQ ID NO: 4962 cttgtcatgcctacgttcc11348 11367 1 3 SEQ ID NO: 3622 aaaatcaagggtgttattt 3473 3492 SEQ ID NO:4963 aaatcttattggggatttt 7084 7103 1 3 SEQ ID NO: 3623tccataccccgtttgcaag 3491 3510 SEQ ID NO: 4964 cttggattcaaaatgtgga 68586877 1 3 SEQ ID NO: 3624 tgcaagcagaagccagaag 3504 3523 SEQ ID NO: 4965cttcagggaacacaatgca 5185 5204 1 3 SEQ ID NO: 3625 cagaagccagaagtgagat3510 3529 SEQ ID NO: 4966 atctatgccatctcttctg 5633 5652 1 3 SEQ ID NO:3626 tgagatcctcgcccactgg 3523 3542 SEQ ID NO: 4967 ccagcttccccacatctca8341 8360 1 3 SEQ ID NO: 3627 ggtcgcctgccaaactgct 3540 3559 SEQ ID NO:4968 agcacatatgaactggacc 13947 13966 1 3 SEQ ID NO: 3628tgcttctccaaatggactc 3555 3574 SEQ ID NO: 4969 gagtttatcagtcagagca 97019720 1 3 SEQ ID NO: 3629 tggactcatctgctacagc 3567 3588 SEQ ID NO: 4970gctgcagtggcccgttcca 8167 8186 1 3 SEQ ID NO: 3630 gctacagcttatggctcca3578 3597 SEQ ID NO: 4971 tggaggacattcctctagc 8211 8230 1 3 SEQ ID NO:3631 ggtggcatggcattatgat 3610 3629 SEQ ID NO: 4972 atcacaaattagtttcacc8947 8966 1 3 SEQ ID NO: 3632 agagaagattgaatttgaa 3631 3650 SEQ ID NO:4973 ttcaacgatacctgtctct 7713 7732 1 3 SEQ ID NO: 3633caggcaccaatgtagatac 3657 3676 SEQ ID NO: 4974 gtatgctaatagactcctg 37363755 1 3 SEQ ID NO: 3634 gacttccaatttccctgtg 3685 3704 SEQ ID NO: 4975cacaatgcaaaattcagtc 5195 5214 1 3 SEQ ID NO: 3635 gtccctcaaacagacatga3764 3783 SEQ ID NO: 4976 tcataagggaggtagggac 12777 12796 1 3 SEQ ID NO:3636 caaacagacatgactttcc 3770 3789 SEQ ID NO: 4977 ggaactacaatttcatttg7022 7041 1 3 SEQ ID NO: 3637 atagttgcaatgagctcat 3809 3828 SEQ ID NO:4978 atgatttgaaaatagctat 6693 6712 1 3 SEQ ID NO: 3638gcttcagaaggcatctggg 3829 3848 SEQ ID NO: 4979 cccaagaggtatttaaagc 1295712976 1 3 SEQ ID NO: 3639 ggagttcaacctccagaac 3895 3914 SEQ ID NO: 4980gttcactccattaacctcc 6314 6333 1 3 SEQ ID NO: 3640 agaaaacctcttcttaaaa3940 3959 SEQ ID NO: 4981 ttttctaaatggaacttct 12173 12192 1 3 SEQ ID NO:3641 aaaacctcttcttaaaaag 3942 3961 SEQ ID NO: 4982 ctttgaaaaattctctttt9213 9232 1 3 SEQ ID NO: 3642 aaaaagcgatggccgggtc 3955 3974 SEQ ID NO:4983 gaccttgcaagaatatttt 6343 6362 1 3 SEQ ID NO: 3643gtcaaatataccttgaaca 3971 3990 SEQ ID NO: 4984 tgttaacaaattccttgac 73537382 1 3 SEQ ID NO: 3644 tgaacaagaacagtttgaa 3984 4003 SEQ ID NO: 4985ttcaagttcctgaccttca 8310 8329 1 3 SEQ ID NO: 3645 agtttgaaaattgagattc3995 4014 SEQ ID NO: 4986 gaatctggctccctcaact 9047 9066 1 3 SEQ ID NO:3646 gtttgaaaattgagattca 3996 4015 SEQ ID NO: 4987 ggaaataccaagtcaaaac10454 10473 1 3 SEQ ID NO: 3647 ttgaaaattgagattcctt 3998 4017 SEQ ID NO:4988 aaggaaaagcgcacctcaa 12030 12049 1 3 SEQ ID NO: 3648ctaaagatgttagagactg 4048 4065 SEQ ID NO: 4989 cagttgaccacaagcttag 1054510564 1 3 SEQ ID NO: 3649 atgttagagactgttagga 4052 4071 SEQ ID NO: 4990tccttaacaccttccacat 8073 8092 1 3 SEQ ID NO: 3650 cagccctccacttcaagtc4074 4093 SEQ ID NO: 4991 gacttctctagtcaggctg 8813 8832 1 3 SEQ ID NO:3651 agccctccacttcaagtct 4075 4094 SEQ ID NO: 4992 agacatcgctgggctggct5728 5747 1 3 SEQ ID NO: 3652 ccatctgccatctcgagag 4102 4121 SEQ ID NO:4993 ctctcaaatgacatgatgg 5330 5349 1 3 SEQ ID NO: 3653attcccaagttgtatcaac 4142 4161 SEQ ID NO: 4994 gttgagaagccccaagaat 62546273 1 3 SEQ ID NO: 3654 tcaactgcaagtgcctctc 4156 4175 SEQ ID NO: 4995gagatcaagacactgttga 8843 8862 1 3 SEQ ID NO: 3655 ggtgttctagacctctcca4178 4197 SEQ ID NO: 4996 tggaaccctctccctcacc 4735 4754 1 3 SEQ ID NO:3656 ctccacgaatgtctacagc 4192 4211 SEQ ID NO: 4997 gctggtaacctaaaaggag5588 5607 1 3 SEQ ID NO: 3657 cacgaatgtctacagcaac 4195 4214 SEQ ID NO:4998 gttgcccaccatcatcgtg 11671 11690 1 3 SEQ ID NO: 3658acgaatgtctacagcaact 4196 4215 SEQ ID NO: 4999 agttgcccaccatcatcgt 1167011689 1 3 SEQ ID NO: 3659 tcctacagtggtggcaaca 4232 4251 SEQ ID NO: 5000tgttagttgctcttaagga 13359 13378 1 3 SEQ ID NO: 3660 cgttaccacatgaaggctg4260 4299 SEQ ID NO: 5001 cagcaagtacctgagaacg 8611 8630 1 3 SEQ ID NO:3661 gaaggctgactctgtggtt 4291 4310 SEQ ID NO: 5002 aacctatgccttaatcttc13169 13188 1 3 SEQ ID NO: 3662 tgtggttgacctgctttcc 4303 4322 SEQ ID NO:5003 ggaaagttaaaacaacaca 6965 6984 1 3 SEQ ID NO: 3663cctgctttcctacaatgtg 4312 4331 SEQ ID NO: 5004 cacaccttgacattgcagg 1108811107 1 3 SEQ ID NO: 3664 ctgctttcctacaatgtgc 4313 4332 SEQ ID NO: 5005gcacaccttgacattgcag 11087 11106 1 3 SEQ ID NO: 3665 tcctacaatgtgcaaggat4319 4338 SEQ ID NO: 5006 atccgctggctctgaagga 8577 8596 1 3 SEQ ID NO:3666 tatgaccacaagaatacgt 4352 4371 SEQ ID NO: 5007 acgtccgtgtgccttcata9984 10003 1 3 SEQ ID NO: 3667 atgaccacaagaatacgtc 4353 4372 SEQ ID NO:5008 gacgtccgtgtgccttcat 9983 10002 1 3 SEQ ID NO: 3668gaatacgtctacactatca 4363 4382 SEQ ID NO: 5009 tgattatctgaattcattc 64876506 1 3 SEQ ID NO: 3669 tttctagattcgaatatca 4406 4425 SEQ ID NO: 5010tgatttacatgatttgaaa 6685 6704 1 3 SEQ ID NO: 3670 gattcgaatatcaaattca4412 4431 SEQ ID NO: 5011 tgaagtagctgagaaaatc 7102 7121 1 3 SEQ ID NO:3671 gaaacaacccagtctcaaa 4449 4468 SEQ ID NO: 5012 tttgaaaaattctcttttc9214 9233 1 3 SEQ ID NO: 3672 cccagtctcaaaaggttta 4456 4475 SEQ ID NO:5013 taaattcattactcctggg 11302 11321 1 3 SEQ ID NO: 3673ctcaaaaggtttactaata 4462 4481 SEQ ID NO: 5014 tattcaaaactgagttgag 1223112250 1 3 SEQ ID NO: 3674 tcaaaaggtttactaatat 4463 4482 SEQ ID NO: 5015atattcaaaactgagttga 12230 12249 1 3 SEQ ID NO: 3675 aaaaggtttactaatattc4465 4484 SEQ ID NO: 5016 gaatttgaaagttcgtttt 9280 9299 1 3 SEQ ID NO:3676 gaaacagcatttgtttgtc 4543 4562 SEQ ID NO: 5017 gacagcatcttcgtgtttc11214 11233 1 3 SEQ ID NO: 3677 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO:5018 acttaaaaaatataaaaat 8022 8041 1 3 SEQ ID NO: 3678tcaagattgatgggcagtt 4569 4588 SEQ ID NO: 5019 aactctcaagtcaagttga 1342213441 1 3 SEQ ID NO: 3679 ttcagagtctcttcgttct 4588 4605 SEQ ID NO: 5020agaagatggcaaatttgaa 11995 12014 1 3 SEQ ID NO: 3680 cagagtctcttcgttctat4588 4607 SEQ ID NO: 5021 atagcatggacttcttctg 8873 8892 1 3 SEQ ID NO:3681 atgctaaaggcacatatgg 4605 4624 SEQ ID NO: 5022 ccatttgagatcacggcat9245 9264 1 3 SEQ ID NO: 3682 gcacatatggcctgtcttg 4614 4633 SEQ ID NO:5023 caagttggcaagtaagtgc 9372 9391 1 3 SEQ ID NO: 3683gagtccaacctgaggttta 4667 4686 SEQ ID NO: 5024 taaagtgccacttttactc 61906209 1 3 SEQ ID NO: 3684 agtccaacctgaggtttaa 4668 4687 SEQ ID NO: 5025ttaacagggaagatagact 9308 9327 1 3 SEQ ID NO: 3685 cctacctccaaggcaccaa4692 4711 SEQ ID NO: 5026 ttggcaagtaagtgctagg 9376 9395 1 3 SEQ ID NO:3686 gaagatggaaccctctccc 4730 4749 SEQ ID NO: 5027 gggaagaagaggcagcttc12291 12310 1 3 SEQ ID NO: 3687 tgatctgcaaagtggcatc 4762 4781 SEQ ID NO:5028 gatgaggaaactcagatca 12263 12282 1 3 SEQ ID NO: 3688gatctgcaaagtggcatca 4763 4782 SEQ ID NO: 5029 tgatgaggaaactcagatc 1226212281 1 3 SEQ ID NO: 3689 gcttccctaaagtatgaga 4793 4812 SEQ ID NO: 5030tctcgtgtctaggaaaagc 5977 5996 1 3 SEQ ID NO: 3690 gtatgagaactacgagctg4804 4823 SEQ ID NO: 5031 cagcttaagagacacatac 6920 6939 1 3 SEQ ID NO:3691 tctaacaagatggatatga 4868 4887 SEQ ID NO: 5032 tcattttccaactaataga13032 13051 1 3 SEQ ID NO: 3692 ctgctgcgttctgaatatc 4907 4926 SEQ ID NO:5033 gatacaagaaaaactgcag 6901 6920 1 3 SEQ ID NO: 3693tcattgaggttcttcagcc 4940 4959 SEQ ID NO: 5034 ggctcatatgctgaaatga 53485367 1 3 SEQ ID NO: 3694 ttctggatcactaaattcc 4953 4982 SEQ ID NO: 5035ggaaggacaaggcccagaa 12549 12568 1 3 SEQ ID NO: 3695 ccatggtcttgagttaaat4981 5000 SEQ ID NO: 5036 atttttattcctgccatgg 10103 10122 1 3 SEQ ID NO:3696 tcttaggcactgacaaaat 5007 5026 SEQ ID NO: 5037 attttttgcaagttaaaga14019 14038 1 3 SEQ ID NO: 3697 acaaggcgacactaaggat 5040 5059 SEQ ID NO:5038 atccatgatctacatttgt 6794 6813 1 3 SEQ ID NO: 3698tgcaacgaccaacttgaag 5083 5102 SEQ ID NO: 5039 cttcagggaacacaatgca 51855204 1 3 SEQ ID NO: 3699 caacttgaagtgtagtctc 5092 5111 SEQ ID NO: 5040gagatgagagatgccgttg 6239 6258 1 3 SEQ ID NO: 3700 gctggagaatgagctgaat5116 5135 SEQ ID NO: 5041 attctcttttcttttcagc 9222 9241 1 3 SEQ ID NO:3701 gcagagcttggcctctctg 5135 5154 SEQ ID NO: 5042 cagatacaagaaaaactgc6899 6918 1 3 SEQ ID NO: 3702 tctctggggcatctatgaa 5148 5167 SEQ ID NO:5043 ttcattcaattgggagaga 6499 6518 1 3 SEQ ID NO: 3703tctggggcatctatgaaat 5150 5169 SEQ ID NO: 5044 atttgtaagaaaatacaga 64366455 1 3 SEQ ID NO: 3704 aacacaatgcaaaattcag 5193 5212 SEQ ID NO: 5045ctgaagcattaaaactgtt 7506 7525 1 3 SEQ ID NO: 3705 ctcacagagctatcactgg5231 5250 SEQ ID NO: 5046 ccagatgctgaacagtgag 8149 8168 1 3 SEQ ID NO:3706 tgggaagtgcttatcaggc 5247 5266 SEQ ID NO: 5047 gcctacgttccatgtccca11356 11375 1 3 SEQ ID NO: 3707 ttcaaggtcagtcaagaag 5303 5322 SEQ ID NO:5048 cttcagtgcagaatatgaa 11977 11996 1 3 SEQ ID NO: 3708aatgacatgatgggctcat 5338 5355 SEQ ID NO: 5049 atgattatctgaattcatt 64866505 1 3 SEQ ID NO: 3709 gctcatatgctgaaatgaa 5349 5368 SEQ ID NO: 5050ttcagccattgacatgagc 5746 5765 1 3 SEQ ID NO: 3710 atatgctgaaatgaaattt5353 5372 SEQ ID NO: 5051 aaatagctattgctaatat 6702 6721 1 3 SEQ ID NO:3711 tctgaacattgcaggctta 5386 5405 SEQ ID NO: 5052 taagaaccagaagatcaga10996 11015 1 3 SEQ ID NO: 3712 gaacattgcaggcttatca 5389 5408 SEQ ID NO:5053 tgatatcgacgtgaggttc 12490 12509 1 3 SEQ ID NO: 3713tgcaggcttatcactggac 5395 5414 SEQ ID NO: 5054 gtcctggattccacatgca 1185211871 1 3 SEQ ID NO: 3714 tcaaaacttgacaacattt 5420 5439 SEQ ID NO: 5055aaattccttgacatgttga 7370 7389 1 3 SEQ ID NO: 3715 atttacagctctgacaagt5435 5454 SEQ ID NO: 5056 acttaaaaaatataaaaat 8022 8041 1 3 SEQ ID NO:3716 ctctgacaagttttataag 5443 5482 SEQ ID NO: 5057 cttacttgaattccaagag10674 10693 1 3 SEQ ID NO: 3717 gttaatttacagctacagc 5488 5487 SEQ ID NO:5058 gctgcatgtggctggtaac 5578 5597 1 3 SEQ ID NO: 3718ttctctggtaactacttta 5491 5510 SEQ ID NO: 5059 taaaagattactttgagaa 72767294 1 3 SEQ ID NO: 3719 cctaaaaggagcctaccaa 5596 5815 SEQ ID NO: 5060ttggcaagtaagtgctagg 9370 9395 1 3 SEQ ID NO: 3720 aaaaggagcctaccaaaat5599 5618 SEQ ID NO: 5061 atttacaattgttgctttt 6271 6290 1 3 SEQ ID NO:3721 aggagcctaccaaaataat 5602 5621 SEQ ID NO: 5062 attacctatgatttctcct10127 10146 1 3 SEQ ID NO: 3722 ataatgaaataaaacacat 5616 5635 SEQ ID NO:5063 atgtcaaacactttgttat 7065 7084 1 3 SEQ ID NO: 3723aaaacacatctatgccatc 5626 5845 SEQ ID NO: 5064 gatgaagatgacgactttt 1215812177 1 3 SEQ ID NO: 3724 tgctaaggttcagggtgtg 5686 5705 SEQ ID NO: 5065cacaagtcgattcccagca 9087 9106 1 3 SEQ ID NO: 3725 gagtttagccatcggctca5705 5724 SEQ ID NO: 5066 tgaggtgactcagagactc 7450 7469 1 3 SEQ ID NO:3726 gctggcttcagccattgac 5740 5759 SEQ ID NO: 5067 gtcagtgaagttctccagc8596 8616 1 3 SEQ ID NO: 3727 atttcagcaatgtcttccg 5790 5809 SEQ ID NO:5068 cggagcatgggagtgaaat 8628 8647 1 3 SEQ ID NO: 3728tttcagcaatgtcttccgt 5791 5810 SEQ ID NO: 5069 acggagcatgggagtgaaa 86278646 1 3 SEQ ID NO: 3729 ttcagcaatgtcttccgtt 5792 5811 SEQ ID NO: 5070aacggagcatgggagtgaa 8628 8645 1 3 SEQ ID NO: 3730 cagcaatgtcttccgttct5794 5813 SEQ ID NO: 5071 agaagtgtcttcaaagctg 12412 12431 1 3 SEQ ID NO:3731 tgtcttccgttctgtaatg 5800 5819 SEQ ID NO: 5072 cattcaattgggagagaca6501 8520 1 3 SEQ ID NO: 3732 gtcttccgttctgtaatgg 5801 5820 SEQ ID NO:5073 ccattcagtctctcaagac 12975 12994 1 3 SEQ ID NO: 3733atgggaaactcgctctctg 5859 5878 SEQ ID NO: 5074 cagataaaaaactcaccat 1221312232 1 3 SEQ ID NO: 3734 ggagaacatactgggcagc 5879 5898 SEQ ID NO: 5075gctgttttgaagactctcc 1088 1107 1 3 SEQ ID NO: 3735 gttgaaagcagaacctctg5914 5933 SEQ ID NO: 5076 cagaattcataatcccaac 8274 8293 1 3 SEQ ID NO:3736 gtctaggaaaagcatcagt 5983 6002 SEQ ID NO: 5077 actgcaagatttttcagac13612 13631 1 3 SEQ ID NO: 3737 agcatcagtgcagctcttg 5993 6012 SEQ ID NO:5078 caagaacctgttagttgct 13351 13370 1 3 SEQ ID NO: 3738ttgaacacaaagtcagtgc 6009 6028 SEQ ID NO: 5079 gcacatcaatattgatcaa 64186437 1 3 SEQ ID NO: 3739 gcagacaggcacctggaaa 6046 6085 SEQ ID NO: 5080tttcagatggcattgctgc 11610 11629 1 3 SEQ ID NO: 3740 gaaactcaagacccaattt6061 6080 SEQ ID NO: 5081 aaatcccatccaggttttc 8037 8056 1 3 SEQ ID NO:3741 acaatgaatacagccagga 6084 6103 SEQ ID NO: 5082 tcctttggctgtgctttgt9682 9701 1 3 SEQ ID NO: 3742 cttggatgcttacaacact 6103 6122 SEQ ID NO:5083 agtgaagttctccagcaag 8599 8618 1 3 SEQ ID NO: 3743ttggcgtggagcttactgg 6132 6151 SEQ ID NO: 5084 ccagaattcataatcccaa 82738292 1 3 SEQ ID NO: 3744 cacttttactcagtgagcc 6198 6217 SEQ ID NO: 5085ggctattgatgttagagtg 6988 7007 1 3 SEQ ID NO: 3745 tttagagatgagagatgcc6235 6254 SEQ ID NO: 5086 ggcatgatgctcatttaaa 9177 9196 1 3 SEQ ID NO:3746 gagaagccccaagaattta 6257 6276 SEQ ID NO: 5087 taaagccattcagtctctc12970 12989 1 3 SEQ ID NO: 3747 caattgttgcttttgtaaa 6276 6295 SEQ ID NO:5088 tttaaccagtcagatattg 10187 10206 1 3 SEQ ID NO: 3748ttttgtaaagtatgataaa 6286 6305 SEQ ID NO: 5089 tttattgctgaatccaaaa 1365513674 1 3 SEQ ID NO: 3749 ttgtaaagtatgataaaaa 6288 6307 SEQ ID NO: 5090ttttgagaggaatcgacaa 6358 6377 1 3 SEQ ID NO: 3750 ttcactccattaacctccc6315 6334 SEQ ID NO: 5091 gggaaaaaacaggcttgaa 9576 9595 1 3 SEQ ID NO:3751 ttttgagaccttgcaagaa 6337 6358 SEQ ID NO: 5092 ttctctctatgggaaaaaa9566 9585 1 3 SEQ ID NO: 3752 accttgcaagaatattttg 6344 6363 SEQ ID NO:5093 caaaagaagcccaagaggt 12948 12967 1 3 SEQ ID NO: 3753tcaatattgatcaatttgt 6423 6442 SEQ ID NO: 5094 acaaagcagattatgttga 1182911848 1 3 SEQ ID NO: 3754 cagagcagccctgggaaaa 6451 6470 SEQ ID NO: 5095ttttcagaccaactctctg 13522 13641 1 3 SEQ ID NO: 3755 cctgggaaaactcccacag6460 6479 SEQ ID NO: 5096 ctgtctctggtcagccagg 7724 7743 1 3 SEQ ID NO:3756 actcccacagcaagctaat 6469 6488 SEQ ID NO: 5097 attacacttcctttcgagt12869 12888 1 3 SEQ ID NO: 3757 aattcattcaattgggaga 6497 6516 SEQ ID NO:5098 tctcttcctccatggaatt 10479 10498 1 3 SEQ ID NO: 3758ttcaattgggagagacaag 6503 6522 SEQ ID NO: 5099 cttggagtgccagtttgaa 1180811827 1 3 SEQ ID NO: 3759 aggagaaactgactgctct 6534 6553 SEQ ID NO: 5100agagcttatgggatttcct 11163 11182 1 3 SEQ ID NO: 3760 actgactgctctcacaaaa6541 6560 SEQ ID NO: 5101 ttttggcaagctatacagt 8380 8399 1 3 SEQ ID NO:3761 gactgctctcacaaaaaag 6544 6563 SEQ ID NO: 5102 ctttgtgagtttatcagtc9695 9714 1 3 SEQ ID NO: 3762 cagacatatatgatacaat 6641 6660 SEQ ID NO:5103 attggatatccaagatctg 1933 1952 1 3 SEQ ID NO: 3763aatttgatcagtatattaa 6657 6676 SEQ ID NO: 5104 ttaaaagaaatcttcaatt 1381513834 1 3 SEQ ID NO: 3764 tatgatttacatgatttga 6683 6702 SEQ ID NO: 5105tcaatgattatatcccata 13128 13147 1 3 SEQ ID NO: 3765 tttgaaaatagctattgct6697 6716 SEQ ID NO: 5106 agcacagaaaaaattcaaa 13864 13883 1 3 SEQ ID NO:3766 ttgaaaatagctattgcta 6698 6717 SEQ ID NO: 5107 tagcacagaaaaaattcaa13863 13882 1 3 SEQ ID NO: 3767 aatagctattgctaatatt 6703 6722 SEQ ID NO:5108 aataaatggagtctttatt 14084 14103 1 3 SEQ ID NO: 3768attattgatgaaatcattg 6719 6738 SEQ ID NO: 5109 caataccagaattcataat 82688287 1 3 SEQ ID NO: 3769 aaagtcttgatgagcacta 6747 6766 SEQ ID NO: 5110tagtgattacacttccttt 12864 12883 1 3 SEQ ID NO: 3770 aagtcttgatgagcactat6748 6767 SEQ ID NO: 5111 atagcaacactaactactt 8769 8788 1 3 SEQ ID NO:3771 ttgatgagcactatcatat 6753 6772 SEQ ID NO: 5112 atatccaagatgagatcaa13101 13120 1 3 SEQ ID NO: 3772 taattttagtaaaaacaat 6777 6796 SEQ ID NO:5113 attgagattccctccatta 11702 11721 1 3 SEQ ID NO: 3773ttttagtaaaaacaatcca 6780 6799 SEQ ID NO: 5114 tggagtgccagtttgaaaa 1181011829 1 3 SEQ ID NO: 3774 acatttgtttattgaaaat 6805 6824 SEQ ID NO: 5115atttcctaaagctggatgt 11175 11194 1 3 SEQ ID NO: 3775 attgattttaacaaaagtg6824 6843 SEQ ID NO: 5116 cactgttccagttgtcaat 9871 9890 1 3 SEQ ID NO:3776 attttaacaaaagtggaag 6826 6847 SEQ ID NO: 5117 cttcaaagacttaaaaaat8014 8033 1 3 SEQ ID NO: 3777 aaatcagaatccagataca 6888 6907 SEQ ID NO:5118 tgtaccataagccatattt 10088 10107 1 3 SEQ ID NO: 3778gaatccagatacaagaaaa 6894 6913 SEQ ID NO: 5119 ttttctaaacttgaaattc 90659084 1 3 SEQ ID NO: 3779 ttaagagacacatacagaa 6924 6943 SEQ ID NO: 5120ttcttaaacattcctttaa 9491 9510 1 3 SEQ ID NO: 3780 atccagcacctagctggaa6950 6969 SEQ ID NO: 5121 ttccaatttccctgtggat 3688 3707 1 3 SEQ ID NO:3781 tgagcatgtcaaacacttt 7060 7079 SEQ ID NO: 5122 aaagtgccacttttactca6191 6210 1 3 SEQ ID NO: 3782 gagcatgtcaaacactttg 7061 7080 SEQ ID NO:5123 caaatgacatgatgggctc 5334 5353 1 3 SEQ ID NO: 3783aaacactttgttataaatc 7070 7089 SEQ ID NO: 5124 gattatatcccatatgttt 1313313152 1 3 SEQ ID NO: 3784 tgagaaaatcaatgccttc 7111 7130 SEQ ID NO: 5125gaaggaaaagcgcacctca 12029 12048 1 3 SEQ ID NO: 3785 tatgaagtagaccaacaaa7160 7179 SEQ ID NO: 5126 tttgtggagggtagtcata 10331 10350 1 3 SEQ ID NO:3786 aagtagaccaacaaatcca 7164 7183 SEQ ID NO: 5127 tggatgaagatgacgactt12156 12175 1 3 SEQ ID NO: 3787 aagttgaaggagactattc 7223 7242 SEQ ID NO:5128 gaataccaatgctgaactt 10168 10187 1 3 SEQ ID NO: 3788acaagttaagataaaagat 7264 7283 SEQ ID NO: 5129 atctaaattcagttcttgt 1133411353 1 3 SEQ ID NO: 3789 aagataaaagattactttg 7271 7290 SEQ ID NO: 5130caaaatagaagggaatctt 2077 2096 1 3 SEQ ID NO: 3790 gattactttgagaaattag7280 7299 SEQ ID NO: 5131 ctaaacttgaaattcaatc 9069 9088 1 3 SEQ ID NO:3791 tgagaaattagttggattt 7288 7307 SEQ ID NO: 5132 aaatccgtgaggtgactca7443 7462 1 3 SEQ ID NO: 3792 aaattagttggatttattg 7292 7311 SEQ ID NO:5133 caattttgagaatgaattt 10419 10438 1 3 SEQ ID NO: 3793tggatttattgatgatgct 7300 7319 SEQ ID NO: 5134 agcatgcctagtttctcca 99539972 1 3 SEQ ID NO: 3794 tcattgaagatgttaacaa 7353 7372 SEQ ID NO: 5135ttgtagatgaaaccaatga 7422 7441 1 3 SEQ ID NO: 3795 cattgaagatgttaacaaa7354 7373 SEQ ID NO: 5136 tttgtagatgaaaccaatg 7421 7440 1 3 SEQ ID NO:3796 attgaagatgttaacaaat 7355 7374 SEQ ID NO: 5137 atttaagtatgatttcaat10495 10514 1 3 SEQ ID NO: 3797 ttgaagatgttaacaaatt 7356 7375 SEQ ID NO:5138 aatttaagtatgatttcaa 10494 10513 1 3 SEQ ID NO: 3798tgaagatgttaacaaattc 7357 7376 SEQ ID NO: 5139 gaatttaagtatgatttca 1049310512 1 3 SEQ ID NO: 3799 acatgttgataaagaaatt 7380 7399 SEQ ID NO: 5140aattccctgaagttgatgt 11487 11506 1 3 SEQ ID NO: 3800 tttgattaccaccagtttg7406 7425 SEQ ID NO: 5141 caaattgaacatccccaaa 8791 8810 1 3 SEQ ID NO:3801 caaaatccgtgaggtgact 7441 7460 SEQ ID NO: 5142 agtccccctaacagatttg7972 7991 1 3 SEQ ID NO: 3802 aaaatccgtgaggtgactc 7442 7461 SEQ ID NO:5143 gagtgaaatgctgtttttt 8638 8657 1 3 SEQ ID NO: 3803aggtgactcagagactcaa 7452 7471 SEQ ID NO: 5144 ttgatgatatctggaacct 1073110750 1 3 SEQ ID NO: 3804 gtgaaattcaggctctgga 7473 7492 SEQ ID NO: 5145tccaatctcctcttttcac 8409 8428 1 3 SEQ ID NO: 3805 gttgcagtgtatctggaaa7547 7566 SEQ ID NO: 5146 tttcaagcaaatgcacaac 8540 8559 1 3 SEQ ID NO:3806 ttaagttcagcatctttgg 7616 7635 SEQ ID NO: 5147 ccaatgctgaactttttaa10173 10192 1 3 SEQ ID NO: 3807 tgaaggccaaattccgaga 7641 7660 SEQ ID NO:5148 tctcctttcttcatcttca 10213 10232 1 3 SEQ ID NO: 3808aatgtatcaaatggacatt 7684 7703 SEQ ID NO: 5149 aatgaagtccggattcatt 1102111040 1 3 SEQ ID NO: 3809 attcagcaggaacttcaac 7700 7719 SEQ ID NO: 5150gttgagaagccccaagaat 6254 6273 1 3 SEQ ID NO: 3810 acctgtctctggtcagcca7722 7741 SEQ ID NO: 5151 tggcaagtaagtgctaggt 9377 9396 1 3 SEQ ID NO:3811 cctgtctctggtcagccag 7723 7742 SEQ ID NO: 5152 ctggacttctctagtcagg8810 8829 1 3 SEQ ID NO: 3812 ggtcagccaggtttatagc 7732 7751 SEQ ID NO:5153 gctaaaggagcagttgacc 10535 10554 1 3 SEQ ID NO: 3813ccaggtttatagcacactt 7738 7757 SEQ ID NO: 5154 aagtccggattcattctgg 1102511044 1 3 SEQ ID NO: 3814 gtttatagcacacttgtca 7742 7761 SEQ ID NO: 5155tgacctgtccattcaaaac 13681 13700 1 3 SEQ ID NO: 3815 acttgtcacctacatttct7753 7772 SEQ ID NO: 5156 agaaaaaggggattgaagt 10283 10302 1 3 SEQ ID NO:3816 ctgattggtggactcttgc 7770 7789 SEQ ID NO: 5157 gcaagttaaagaaaatcag14026 14045 1 3 SEQ ID NO: 3817 atgaaagcattggtagagc 7847 7866 SEQ ID NO:5158 gctcatctcctttcttcat 10208 10227 1 3 SEQ ID NO: 3818tgaaagcattggtagagca 7848 7867 SEQ ID NO: 5159 tgctcatctcctttcttca 1020710226 1 3 SEQ ID NO: 3819 gggttcactgttcctgaaa 7868 7887 SEQ ID NO: 5160tttcaccatagaaggaccc 8959 8978 1 3 SEQ ID NO: 3820 tcaagaccatccttgggac7887 7906 SEQ ID NO: 5161 gtccccctaacagatttga 7973 7992 1 3 SEQ ID NO:3821 ccttgggaccatgcctgcc 7897 7916 SEQ ID NO: 5162 ggcaccagggctcggaagg13978 13997 1 3 SEQ ID NO: 3822 ttcaggctcttcagaaagc 7929 7948 SEQ ID NO:5163 gcttgaaggaattcttgaa 9588 9607 1 3 SEQ ID NO: 3823ttcagataaacttcaaaga 8004 8023 SEQ ID NO: 5164 tcttcataagttcaatgaa 1318313202 1 3 SEQ ID NO: 3824 acttcaaagacttaaaaaa 8013 8032 SEQ ID NO: 5165ttttaacaaaagtggaagt 6829 6848 1 3 SEQ ID NO: 3825 atcccatccaggttttcca8039 8058 SEQ ID NO: 5166 tggagaagcaaatctggat 9472 9491 1 3 SEQ ID NO:3826 gaatttaccatccttaaca 8063 8082 SEQ ID NO: 5167 tgttgaagtgtctccattc9889 9908 1 3 SEQ ID NO: 3827 cattccttcctttacaatt 8089 8108 SEQ ID NO:5168 aattccaattttgagaatg 10414 10433 1 3 SEQ ID NO: 3828ttgaccagatgctgaacag 8145 8164 SEQ ID NO: 5169 ctgttgaaagatttatcaa 1293212951 1 3 SEQ ID NO: 3829 aatcaccctgccagacttc 8233 8252 SEQ ID NO: 5170gaagttctcaattttgatt 8522 8541 1 3 SEQ ID NO: 3830 tgaccttcacataccagaa8320 8339 SEQ ID NO: 5171 ttcttctggaaaagggtca 8884 8903 1 3 SEQ ID NO:3831 ttccagcttccccacatct 8339 8358 SEQ ID NO: 5172 agattctcagatgagggaa8921 8940 1 3 SEQ ID NO: 3832 aagctatacagtattctga 8387 8406 SEQ ID NO:5173 tcagatggcattgctgctt 11612 11631 1 3 SEQ ID NO: 3833attctgaaaatccaatctc 8399 8418 SEQ ID NO: 5174 gagataaccgtgcctgaat 1155211571 1 3 SEQ ID NO: 3834 tttcacattagatgcaaat 8422 8441 SEQ ID NO: 5175attttgaaaaaaacagaaa 9738 9757 1 3 SEQ ID NO: 3835 caaatgctgacatagggaa8436 8455 SEQ ID NO: 5176 ttccatcacaaatcctttg 9670 9689 1 3 SEQ ID NO:3836 gagagtccaaattagaagt 8508 8527 SEQ ID NO: 5177 actttacttcccaactctc13410 13429 1 3 SEQ ID NO: 3837 agagtccaaattagaagtt 8509 8528 SEQ ID NO:5178 aactttacttcccaactct 13409 13428 1 3 SEQ ID NO: 3838tctcaattttgattttcaa 8527 8546 SEQ ID NO: 5179 ttgattcccttttttgaga 1153711556 1 3 SEQ ID NO: 3839 caattttgattttcaagca 8530 8549 SEQ ID NO: 5180tgctgaatccaaaagattg 13660 13679 1 3 SEQ ID NO: 3840 aatgcacaactctcaaacc8549 8568 SEQ ID NO: 5181 ggtttatcaaggggccatt 12460 12479 1 3 SEQ ID NO:3841 agttctccagcaagtacct 8604 8623 SEQ ID NO: 5182 aggttccatcgtgcaaact11388 11407 1 3 SEQ ID NO: 3842 agtacctgagaacggagca 8616 8635 SEQ ID NO:5183 tgctccaggagaacttact 13780 13799 1 3 SEQ ID NO: 3843tcaaacacagtggcaagtt 8678 8697 SEQ ID NO: 5184 aactctcaagtcaagttga 1342213441 1 3 SEQ ID NO: 3844 acaatcagcttaccctgga 8751 8770 SEQ ID NO: 5185tccattctgaatatattgt 13380 13399 1 3 SEQ ID NO: 3845 ctggatagcaacactaaat8765 8784 SEQ ID NO: 5186 attttctgaacttccccag 12702 12721 1 3 SEQ ID NO:3846 ctgacctgcgcaacgagat 8829 8848 SEQ ID NO: 5187 atctgatgaggaaactcag12259 12278 1 3 SEQ ID NO: 3847 agatgagggaacacatgaa 8929 8948 SEQ ID NO:5188 ttcatgtccctagaaatct 10038 10057 1 3 SEQ ID NO: 3848tcaacttttctaaacttga 9060 9079 SEQ ID NO: 5189 tcaaggataacgtgtttga 1261812637 1 3 SEQ ID NO: 3849 ttctaaacttgaaattcaa 9067 9086 SEQ ID NO: 5190ttgatgatgctgtcaagaa 7308 7327 1 3 SEQ ID NO: 3850 gaaattcaatcacaagtcg9077 9096 SEQ ID NO: 5191 cgacgaagaaaataatttc 13566 13585 1 3 SEQ ID NO:3851 cactgtttggagaagggaa 9141 9160 SEQ ID NO: 5192 ttccagaaagcagccagtg12506 12525 1 3 SEQ ID NO: 3852 actgtttggagaagggaag 9142 9161 SEQ ID NO:5193 cttccccaaagagaccagt 2898 2917 1 3 SEQ ID NO: 3853aattctcttttcttttcag 9221 9240 SEQ ID NO: 5194 ctgattactatgaaaaatt 1363813657 1 3 SEQ ID NO: 3854 ttcttttcagcccagccat 9230 9249 SEQ ID NO: 5195atggaaaagggaaagagaa 13494 13513 1 3 SEQ ID NO: 3855 tttgaaagttcgttttcca9283 9302 SEQ ID NO: 5196 tggaagtgtcagtggcaaa 10380 10399 1 3 SEQ ID NO:3856 cagggaagatagacttcct 9312 9331 SEQ ID NO: 5197 aggacctttcaaattcctg9848 9867 1 3 SEQ ID NO: 3857 ataagtacaaccaaaattt 9405 9424 SEQ ID NO:5198 aaatcaggatctgagttat 14038 14057 1 3 SEQ ID NO: 3858acaacgagaacattatgga 9435 9454 SEQ ID NO: 5199 tccattctgaatatattgt 1338013399 1 3 SEQ ID NO: 3859 aggaataaatggagaagca 9463 9482 SEQ ID NO: 5200tgctggaattgtcattcct 11734 11753 1 3 SEQ ID NO: 3860 agcaaatctggatttctta9478 9497 SEQ ID NO: 5201 taagttctctgtacctgct 11719 11738 1 3 SEQ ID NO:3861 tcctttaacaattcctgaa 9502 9521 SEQ ID NO: 5202 ttcaaaacgagcttcagga13206 13225 1 3 SEQ ID NO: 3862 tttaacaattcctgaaatg 9505 9524 SEQ ID NO:5203 catttgatttaagtgtaaa 9621 9640 1 3 SEQ ID NO: 3863acacaataatcacaactcc 9534 9553 SEQ ID NO: 5204 ggagacagcatcttcgtgt 1121111230 1 3 SEQ ID NO: 3864 aagatttctctctatggga 9561 9580 SEQ ID NO: 5205tcccagaaaacctcttctt 3936 3955 1 3 SEQ ID NO: 3865 gaaaaaacaggcttgaagg9578 9597 SEQ ID NO: 5206 ccttttacaattcattttc 13021 13040 1 3 SEQ ID NO:3866 ttgaaggaattcttgaaaa 9590 9609 SEQ ID NO: 5207 ttttgagaatgaatttcaa10422 10441 1 3 SEQ ID NO: 3867 tgaaggaattcttgaaaac 9591 9610 SEQ ID NO:5208 gttttggctgataaattca 11291 11310 1 3 SEQ ID NO: 3868agctcagtataagaaaaac 9640 9659 SEQ ID NO: 5209 gtttgataagtacaaagct 98059824 1 3 SEQ ID NO: 3869 tcaaatcctttgacaggca 9720 9739 SEQ ID NO: 5210tgcctgagcagaccattga 11688 11707 1 3 SEQ ID NO: 3870 atgaaacaaaaattaagtt9789 9808 SEQ ID NO: 5211 aactttgcactatgttcat 12762 12781 1 3 SEQ ID NO:3871 aattcctggatacactgtt 9859 9878 SEQ ID NO: 5212 aacacatgaatcacaaatt8938 8957 1 3 SEQ ID NO: 3872 ttccagttgtcaatgttga 9876 9895 SEQ ID NO:5213 tcaaaacgagcttcaggaa 13207 13226 1 3 SEQ ID NO: 3873aagtgtctccattcaccat 9894 9913 SEQ ID NO: 5214 atgggaagtataagaactt 48424861 1 3 SEQ ID NO: 3874 gtcagcatgcctagtttct 9950 9969 SEQ ID NO: 5215agaaaaggcacaccttgac 11080 11099 1 3 SEQ ID NO: 3875 ctgccatgggcaatattac10113 10132 SEQ ID NO: 5216 gtaagaaaatacagagcag 6440 6459 1 3 SEQ ID NO:3876 tgaataccaatgctgaact 10167 10186 SEQ ID NO: 5217 agttgaaggagactattca7224 7243 1 3 SEQ ID NO: 3877 tattgttgctcatctcctt 10201 10220 SEQ ID NO:5218 aaggaaacataaactaata 12889 12908 1 3 SEQ ID NO: 3878tgttgctcatctcctttct 10204 10223 SEQ ID NO: 5219 agaagaaatctgcagaaca12431 12450 1 3 SEQ ID NO: 3879 tctgtcattgatgcactgc 10232 10251 SEQ IDNO: 5220 gcagtagactataagcaga 13928 13947 1 3 SEQ ID NO: 3880ccacagctctgtctctgag 10305 10324 SEQ ID NO: 5221 ctcagggatctgaaggtgg 81958214 1 3 SEQ ID NO: 3881 atttgtggagggtagtcat 10330 10349 SEQ ID NO: 5222atgaagtagaccaacaaat 7161 7180 1 3 SEQ ID NO: 3882 atatggaagtgtcagtggc10377 10396 SEQ ID NO: 5223 gccacactccaacgcatat 10778 10797 1 3 SEQ IDNO: 3883 tggaaataccaagtcaaaa 10453 10472 SEQ ID NO: 5224ttttacaattcattttcca 13023 13042 1 3 SEQ ID NO: 3884 aagtcaaaacctactgtct10463 10482 SEQ ID NO: 5225 agacctagtgattacactt 12859 12878 1 3 SEQ IDNO: 3885 actgtctcttcctccatgg 10475 10494 SEQ ID NO: 5226ccatgcaagtcagcccagt 10924 10943 1 3 SEQ ID NO: 3886 cttcctccatggaatttaa10482 10501 SEQ ID NO: 5227 ttaatcgagaggtatgaag 7148 7167 1 3 SEQ ID NO:3887 attcttcaatgctgtactc 10512 10531 SEQ ID NO: 5228 gagttgagggtccgggaat12242 12261 1 3 SEQ ID NO: 3888 ttgaccacaagcttagctt 10548 10567 SEQ IDNO: 5231 aagcgcacctcaatatcaa 12036 12055 1 3 SEQ ID NO: 3889cctcacctcttacttttcc 10573 10592 SEQ ID NO: 5232 ggaactattgctagtgagg10649 10668 1 3 SEQ ID NO: 3890 agctgcagggcacttccaa 10710 10729 SEQ IDNO: 5233 ttgggaagaagaggcagct 12289 12308 1 3 SEQ ID NO: 3891ttccaaaattgatgatatc 10723 10742 SEQ ID NO: 5234 gatatacactagggaggaa12745 12764 1 3 SEQ ID NO: 3892 gagaacatacaagcaaagc 10860 10879 SEQ IDNO: 5235 gcttggttttgccagtctc 2467 2486 1 3 SEQ ID NO: 3893atggcaaatgtcagctctt 10897 10916 SEQ ID NO: 5236 aagaggtatttaaagccat12960 12979 1 3 SEQ ID NO: 3894 tggcaaatgtcagctcttg 10898 10917 SEQ IDNO: 5237 caagaggtatttaaagcca 12959 12978 1 3 SEQ ID NO: 3895ttgttcaggtccatgcaag 10914 10933 SEQ ID NO: 5238 cttgggggaggaggaacaa14066 14085 1 3 SEQ ID NO: 3896 tgttcaggtccatgcaagt 10915 10934 SEQ IDNO: 5239 acttgggggaggaggaaca 14065 14084 1 3 SEQ ID NO: 3897agttccttccatgatttcc 10940 10959 SEQ ID NO: 5240 ggaatctgatgaggaaact12256 12275 1 3 SEQ ID NO: 3898 tgctaacactaagaaccag 10987 11006 SEQ IDNO: 5241 ctggatgtaaccaccagca 11186 11205 1 3 SEQ ID NO: 3899actaagaaccagaagatca 10994 11013 SEQ ID NO: 5242 tgatcaagaacctgttagt13347 13366 1 3 SEQ ID NO: 3900 ctaagaaccagaagatcag 10995 11014 SEQ IDNO: 5243 ctgatcaagaacctgttag 13346 13365 1 3 SEQ ID NO: 3901cagaagatcagatggaaaa 11003 11022 SEQ ID NO: 5244 ttttcagaccaactctctg13622 13641 1 3 SEQ ID NO: 3902 aaaaatgaagtccggattc 11018 11037 SEQ IDNO: 5245 gaatttgaaagttcgtttt 9280 9299 1 3 SEQ ID NO: 3903gattcattctgggtctttc 11032 11051 SEQ ID NO: 5246 gaaaacctatgccttaatc13166 13185 1 3 SEQ ID NO: 3904 aagaaaaggcacaccttga 11079 11095 SEQ IDNO: 5247 tcaaaacctactgtctctt 10466 10485 1 3 SEQ ID NO: 3905aaggacacctaaggttcct 11115 11134 SEQ ID NO: 5248 aggacaccaaaataacctt 75727591 1 3 SEQ ID NO: 3906 ccagcattggtaggagaca 11199 11218 SEQ ID NO: 5249tgtcaacaagtaccactgg 12370 12389 1 3 SEQ ID NO: 3907 ctttgtgtacaccaaaaac11239 11258 SEQ ID NO: 5250 gtttttaaattgttgaaag 13148 13167 1 3 SEQ IDNO: 3908 ccatccctgtaaaagtttt 11277 11296 SEQ ID NO: 5251aaaagggtcatggaaatgg 8893 8912 1 3 SEQ ID NO: 3909 tgatctaaattcagttctt11332 11351 SEQ ID NO: 5252 aagatagtcagtctgatca 13334 13353 1 3 SEQ IDNO: 3910 aagaagctgagaacttcat 11432 11451 SEQ ID NO: 5253atgagatcaacacaatctt 13110 13129 1 3 SEQ ID NO: 3911 tttgccctcaacctaccaa11453 11472 SEQ ID NO: 5254 ttggtacgagttactcaaa 12641 12660 1 3 SEQ IDNO: 3912 cttgattcccttttttgag 11536 11555 SEQ ID NO: 5255ctcaattttgattttcaag 8628 8547 1 3 SEQ ID NO: 3913 ttcacgcttccaaaaagtg11591 11610 SEQ ID NO: 5256 cactcattgattttctgaa 12693 12712 1 3 SEQ IDNO: 3914 tgtttcagatggcattgct 11608 11627 SEQ ID NO: 5257agcagattatgttgaaaca 11833 11852 1 3 SEQ ID NO: 3915 aatgcagtagccaacaaga11639 11658 SEQ ID NO: 5258 tcttttcagcccagccatt 9231 9250 1 3 SEQ ID NO:3916 ctgagcagaccattgagat 11691 11710 SEQ ID NO: 5259 atctgatgaggaaactcag12259 12278 1 3 SEQ ID NO: 3917 tgagcagaccattgagatt 11692 11711 SEQ IDNO: 5260 aatctgatgaggaaactca 12258 12277 1 3 SEQ ID NO: 3918ttgagattccctccattaa 11703 11722 SEQ ID NO: 5261 ttaatcttcataagttcaa13179 13198 1 3 SEQ ID NO: 3919 acttggagtgccagtttga 11807 11826 SEQ IDNO: 5262 tcaattgggagagacaagt 6504 6523 1 3 SEQ ID NO: 3920caaatttgaaggacttcag 12004 12023 SEQ ID NO: 5263 ctgagaacttcatcatttg11438 11457 1 3 SEQ ID NO: 3921 agcccagcgttcaccgatc 12056 12075 SEQ IDNO: 5264 gatccaagtatagttggct 13286 13305 1 3 SEQ ID NO: 3922cagcgttcaccgatctcca 12060 12079 SEQ ID NO: 5265 tggacctgcaccaaagctg13960 13979 1 3 SEQ ID NO: 3923 ctccatctgcgctaccaga 12074 12093 SEQ IDNO: 5266 tctgatatacatcacggag 13711 13730 1 3 SEQ ID NO: 3924atgaggaaactcagatcaa 12264 12283 SEQ ID NO: 5267 ttgagttgcccaccatcat11667 11686 1 3 SEQ ID NO: 3925 aggcagcttctggcttgct 12300 12319 SEQ IDNO: 5268 agcaagtctttcctggcct 3018 3037 1 3 SEQ ID NO: 3926tgaaagacaacgtgcccaa 12327 12346 SEQ ID NO: 5269 ttgggagagacaagtttca 65086527 1 3 SEQ ID NO: 3927 tatgattatgtcaacaagt 12362 12381 SEQ ID NO: 5270actttgcactatgttcata 12763 12782 1 3 SEQ ID NO: 3928 cattaggcaaattgatgat12475 12494 SEQ ID NO: 5271 atcaacacaatcttcaatg 13115 13134 1 3 SEQ IDNO: 3929 ttgactcaggaaggccaag 12584 12603 SEQ ID NO: 5272cttggtacgagttactcaa 12640 12659 1 3 SEQ ID NO: 3930 gaaacctgggatatacact12736 12755 SEQ ID NO: 5273 agtgattacacttcctttc 12865 12884 1 3 SEQ IDNO: 3931 tcctttcgagttaaggaaa 12877 12896 SEQ ID NO: 5274tttctgccactgctcagga 13524 13543 1 3 SEQ ID NO: 3932 gccattcagtctctcaaga12974 12993 SEQ ID NO: 5275 tcttccgttctgtaatggc 5802 5821 1 3 SEQ ID NO:3933 gtgctacgtaatcttcagg 13001 13020 SEQ ID NO: 5276 cctgcaccaaagctggcac13964 13983 1 3 SEQ ID NO: 3934 agctgaaagagatgaaatt 13065 13084 SEQ IDNO: 5277 aatttattcaaaacgagct 13200 13219 1 3 SEQ ID NO: 3935aatttacttatcttattaa 13080 13099 SEQ ID NO: 5278 ttaaaagaaatcttcaatt13815 13834 1 3 SEQ ID NO: 3936 ttttaaattgttgaaagaa 13150 13169 SEQ IDNO: 5279 ttctctctatgggaaaaaa 9566 9585 1 3 SEQ ID NO: 3937taatcttcataagttcaat 13180 13199 SEQ ID NO: 5280 attgagattccctccatta11702 11721 1 3 SEQ ID NO: 3938 atattttgatccaagtata 13279 13298 SEQ IDNO: 5281 tataagcagaagcacatat 13937 13956 1 3 SEQ ID NO: 3939tgaaatattatgaacttga 13311 13330 SEQ ID NO: 5282 tcaaccttaatgattttca 82958314 1 3 SEQ ID NO: 3940 caatttctgcacagaaata 13442 13461 SEQ ID NO: 5283tattcttcttttccaattg 13834 13853 1 3 SEQ ID NO: 3941 agaagattgcagagctttc13509 13528 SEQ ID NO: 5284 gaaatcttcaatttattct 13821 13840 1 3 SEQ IDNO: 3942 gaagaaaataatttctgat 13570 13589 SEQ ID NO: 5285atcagttcagataaacttc 7999 8018 1 3 SEQ ID NO: 3943 ttgacctgtccattcaaaa13680 13699 SEQ ID NO: 5286 ttttgagaatgaatttcaa 10422 10441 1 3 SEQ IDNO: 3944 tcaaaactaccacacattt 13693 13712 SEQ ID NO: 5287aaattccttgacatgttga 7370 7389 1 3 SEQ ID NO: 3945 ttttttaaaagaaatcttc13811 13830 SEQ ID NO: 5288 gaagtgtcagtggcaaaaa 10382 10401 1 3 SEQ IDNO: 3946 aggatctgagttattttgc 14043 14062 SEQ ID NO: 5289gcaagggttcactgttcct 7864 7883 1 3 SEQ ID NO: 3947 tttgctaaacttgggggag14057 14076 SEQ ID NO: 5290 ctccccaggacctttcaaa 9842 9861 1 3

TABLE 10 Selected palindromic sequences from human glucose-6-phosphataseStart End Start End Source Index Index Match Index Index # B SEQ ID NO:5291 tccatcttcaggaagctgt 222 241 SEQ ID NO: 5369 acagactctttcagatgga1340 1359 1 6 SEQ ID NO: 5292 ccatcttcaggaagctgtg 223 242 SEQ ID NO:5370 cacagactctttcagatgg 1339 1358 1 6 SEQ ID NO: 5293cctctggccatgccatggg 417 436 SEQ ID NO: 5371 cccattttgaggccagagg 14921511 1 6 SEQ ID NO: 5294 ctctggccatgccatgggc 418 437 SEQ ID NO: 5372gcccattttgaggccagag 1491 1510 1 6 SEQ ID NO: 5295 ttgaatgtcattttgtggt521 540 SEQ ID NO: 5373 accatacattatcattcaa 2945 2964 1 6 SEQ ID NO:5296 tcagtaatgggggaccagc 1886 1905 SEQ ID NO: 5374 gctggtctcgaactcctga2731 2750 1 6 SEQ ID NO: 5297 ttttactgtgcatacatgt 1956 1975 SEQ ID NO:5375 acatctttgaaaagaaaaa 2983 3002 1 6 SEQ ID NO: 5298tgaggtgccaaggaaatga 50 69 SEQ ID NO: 5376 tcatgtctcagcctcctca 2620 26391 5 SEQ ID NO: 5299 gaggtgccaaggaaatgag 51 70 SEQ ID NO: 5377ctcatgtctcagcctcctc 2619 2638 1 5 SEQ ID NO: 5300 gggaaagataaagccgacc487 506 SEQ ID NO: 5378 ggtcgcctggcttattccc 1295 1314 1 5 SEQ ID NO:5301 ttttcctcatcaagttgtt 598 617 SEQ ID NO: 5379 aacatctttgaaaagaaaa2982 3001 1 5 SEQ ID NO: 5302 ctttcagccacatccacag 651 670 SEQ ID NO:5380 ctgtggactctggagaaag 773 792 1 5 SEQ ID NO: 5303 tggactctggagaaagccc776 795 SEQ ID NO: 5381 gggctggctctcaactcca 884 903 1 5 SEQ ID NO: 5304agcctcctcaagaacctgg 848 867 SEQ ID NO: 5382 ccagattcttccactggct 21072126 1 5 SEQ ID NO: 5305 ggcctggggctggctctca 878 897 SEQ ID NO: 5383tgagccaccgcaccgggcc 2801 2820 1 5 SEQ ID NO: 5306 gagctcactcccactggaa1439 1458 SEQ ID NO: 5384 ttccaggtagggccagctc 1676 1695 1 5 SEQ ID NO:5307 agctaatgaagctattgag 1572 1591 SEQ ID NO: 5385 ctcagcctcctcagtagct2626 2645 1 5 SEQ ID NO: 5308 gctaatgaagctattgaga 1573 1592 SEQ ID NO:5386 tctcagcctcctcagtagc 2625 2644 1 5 SEQ ID NO: 5309ctaaatggctttaattata 1854 1873 SEQ ID NO: 5387 tatatttttagaattttag 26832702 1 5 SEQ ID NO: 5310 ctgcttttctttttttttc 2509 2528 SEQ ID NO: 5388gaaaaatatatatgtgcag 2996 3015 1 5 SEQ ID NO: 5311 caatcaccaccaagcctgg 019 SEQ ID NO: 5389 ccagaatgggtccacattg 812 831 1 4 SEQ ID NO: 5312agcctggaataactgcaag 12 31 SEQ ID NO: 5390 cttggatttctgaatggct 1987 20061 4 SEQ ID NO: 5313 gttccatcttcaggaagct 220 239 SEQ ID NO: 5391agctcactcccactggaac 1440 1459 1 4 SEQ ID NO: 5314 tggtgggttttggatactg326 345 SEQ ID NO: 5392 cagtcctcccaccctacca 2425 2444 1 4 SEQ ID NO:5315 acctgtgagactggaccag 392 411 SEQ ID NO: 5393 ctggagaaagcccagaggt 782801 1 4 SEQ ID NO: 5316 gctgttacagaaactttca 638 657 SEQ ID NO: 5394tgaatggtcttctgccagc 1474 1493 1 4 SEQ ID NO: 5317 acagcatctataatgccag666 685 SEQ ID NO: 5395 ctgggtgtagacctcctgt 758 777 1 4 SEQ ID NO: 5318gggtgtagacctcctgtgg 760 779 SEQ ID NO: 5396 ccacattgacaccacaccc 823 8421 4 SEQ ID NO: 5319 ggtgtagacctcctgtgga 761 780 SEQ ID NO: 5397tccacattgacaccacacc 822 841 1 4 SEQ ID NO: 5320 gtgtagacctcctgtggac 762781 SEQ ID NO: 5398 gtccacattgacaccacac 821 840 1 4 SEQ ID NO: 5321gacctcctgtggactctgg 767 786 SEQ ID NO: 5399 ccagatattgcactaggtc 20142033 1 4 SEQ ID NO: 5322 cctgggcacgctctttggc 862 881 SEQ ID NO: 5400gccagctcacaagcccagg 1687 1706 1 4 SEQ ID NO: 5323 ctgggcacgctctttggcc863 882 SEQ ID NO: 5401 ggccagctcacaagcccag 1686 1705 1 4 SEQ ID NO:5324 ctggtcttctacgtcttgt 1028 1047 SEQ ID NO: 5402 acaaaagcaagacttccag1663 1682 1 4 SEQ ID NO: 5325 agagtgcggtagtgcccct 1056 1075 SEQ ID NO:5403 agggccaggattcctctct 2229 2248 1 4 SEQ ID NO: 5326tgggcactggtatttggag 1217 1236 SEQ ID NO: 5404 ctcccactggaacagccca 14461465 1 4 SEQ ID NO: 5327 gaattaaatcacggatggc 1267 1286 SEQ ID NO: 5405gccaaccaagagcacattc 2311 2330 1 4 SEQ ID NO: 5328 tgttgctagaagttgggtt1598 1617 SEQ ID NO: 5406 aaccatcctgctcataaca 2967 2986 1 4 SEQ ID NO:5329 aggagctctgaatctgata 1764 1783 SEQ ID NO: 5407 tatcacattacatcatcct2063 2082 1 4 SEQ ID NO: 5330 taaatggctttaattatat 1855 1874 SEQ ID NO:5408 atatatgtgcagtatttta 3003 3022 1 4 SEQ ID NO: 5331aaaatgacaaggggagggc 2215 2234 SEQ ID NO: 5409 gccctccttgcctgttttt 28172836 1 4 SEQ ID NO: 5332 ttaaaggaaaagtcaacat 2330 2349 SEQ ID NO: 5410atgtgcagtattttattaa 3007 3026 1 4 SEQ ID NO: 5333 acatcttctctcttttttt2345 2364 SEQ ID NO: 5411 aaaagaaaaatatatatgt 2992 3011 1 4 SEQ ID NO:5334 ttctacgtcctcttcccca 197 216 SEQ ID NO: 5412 tgggccagccgcacaagaa1116 1135 1 3 SEQ ID NO: 5335 tgggtagctgtgattggag 257 276 SEQ ID NO:5413 ctcccactggaacagccca 1446 1465 1 3 SEQ ID NO: 5336gctgtgattggagactggc 263 282 SEQ ID NO: 5414 gccatgccatgggcacagc 423 4421 3 SEQ ID NO: 5337 cacttccgtgcccctgata 358 377 SEQ ID NO: 5415tatcacccaggctggagtg 2548 2567 1 3 SEQ ID NO: 5338 acatctactctttccatct464 483 SEQ ID NO: 5416 agatgggatttcatcatgt 2705 2724 1 3 SEQ ID NO:5339 ctactctttccatctttca 468 487 SEQ ID NO: 5417 tgaatactctcacaagtag1419 1438 1 3 SEQ ID NO: 5340 agataaagccgacctacag 492 511 SEQ ID NO:5418 ctgtttttcaatctcatct 2828 2847 1 3 SEQ ID NO: 5341tgtgcagctgaatgtctgt 553 572 SEQ ID NO: 5419 acagaaactttcagccaca 644 6631 3 SEQ ID NO: 5342 atgtctgtctgtcacgaat 564 583 SEQ ID NO: 5420attcaggtatagctgacat 2038 2057 1 3 SEQ ID NO: 5343 ctgtcacgaatctaccttg572 591 SEQ ID NO: 5421 caaggtgctaggattacag 2779 2798 1 3 SEQ ID NO:5344 atcaagttgttgctggagt 606 625 SEQ ID NO: 5422 actcctgacctcaagtgat2742 2761 1 3 SEQ ID NO: 5345 cagaaactttcagccacat 645 664 SEQ ID NO:5423 atgtttcaattaggctctg 2185 2204 1 3 SEQ ID NO: 5346actttcagccacatccaca 650 669 SEQ ID NO: 5424 tgtggcgtatcatgcaagt 18181837 1 3 SEQ ID NO: 5347 atgccagcctcaagaaata 678 697 SEQ ID NO: 5425tattttttttactgtgcat 1950 1969 1 3 SEQ ID NO: 5348 agaaatattttctcattac690 709 SEQ ID NO: 5426 gtaaatatgactcctttct 2283 2302 1 3 SEQ ID NO:5349 gaaatattttctcattacc 691 710 SEQ ID NO: 5427 ggtaaatatgactcctttc2282 2301 1 3 SEQ ID NO: 5350 tgctgctcaagggactggg 744 763 SEQ ID NO:5428 cccaagccaaccaagagca 2306 2325 1 3 SEQ ID NO: 5351cctgtggactctggagaaa 772 791 SEQ ID NO: 5429 tttcatcatgttggccagg 27132732 1 3 SEQ ID NO: 5352 ggagaaagcccagaggtgg 784 803 SEQ ID NO: 5430ccaccgcaccgggccctcc 2805 2824 1 3 SEQ ID NO: 5353 ttgaaacccccatcccaag1004 1023 SEQ ID NO: 5431 cttgaattcctgggctcaa 2405 2424 1 3 SEQ ID NO:5354 cagatggaggtgccatatc 1351 1370 SEQ ID NO: 5432 gatatgcagagtatttctg2847 2866 1 3 SEQ ID NO: 5355 ggagctcactcccactgga 1438 1457 SEQ ID NO:5433 tccacctgccttggcctcc 2760 2779 1 3 SEQ ID NO: 5356ttgggtaatgtttttgaaa 1553 1572 SEQ ID NO: 5434 tttctctatcccaagccaa 22972316 1 3 SEQ ID NO: 5357 gaagttgggttgttctgga 1606 1625 SE0 ID NO: 5435tccaccccactggatcttc 2131 2150 1 3 SEQ ID NO: 5358 aaaagaaggctgcctaagg1785 1804 SEQ ID NO: 5436 ccttgcctgcttttctttt 2503 2522 1 3 SEQ ID NO:5359 aaagaaggctgcctaagga 1786 1805 SEQ ID NO: 5437 tccttgcctgcttttcttt2502 2521 1 3 SEQ ID NO: 5360 aagaaggctgcctaaggag 1787 1806 SEQ ID NO:5438 ctccttgcctgcttttctt 2501 2520 1 3 SEQ ID NO: 5361agaaggctgcctaaggagg 1788 1807 SEQ ID NO: 5439 cctccttgcctgcttttct 25002519 1 3 SEQ ID NO: 5362 atttccttggatttctgaa 1982 2001 SEQ ID NO: 5440ttcaattaggctctgaaat 2189 2208 1 3 SEQ ID NO: 5363 tccttataagcccagctct2081 2100 SEQ ID NO: 5441 agagcacattcttaaagga 2319 2338 1 3 SEQ ID NO:5364 ataagcccagctctgcttt 2086 2105 SEQ ID NO: 5442 aaagctgaagcctatttat2889 2908 1 3 SEQ ID NO: 5365 ggccaggattcctctctca 2231 2250 SEQ ID NO:5443 tgagccaccgcaccgggcc 2801 2820 1 3 SEQ ID NO: 5366gccaactcctccttgcctg 2493 2512 SEQ ID NO: 5444 caggctggagtggagtggc 25552574 1 3 SEQ ID NO: 5367 ttttttttctttttttgag 2519 2538 SEQ ID NO: 5445ctcataacatctttgaaaa 2977 2996 1 3 SEQ ID NO: 5368 ccggcgtgcaccaccatgc2652 2671 SEQ ID NO: 5446 gcatgagccaccgcaccgg 2798 2817 1 3

TABLE 11 Selected palindromic sequences from rat glucose-6-phosphataseStart End Start End Source Index Index Match Index Index # B SEQ ID NO:5447 ctgactattacagcaacag 301 320 SEQ ID NO: 5471 ctgtggctgaaactttcag 598617 1 6 SEQ ID NO: 5448 ctcttggggttggggctgg 831 850 SEQ ID NO: 5472ccagcatgtaccgcaagag 859 878 1 6 SEQ ID NO: 5449 tgcaaaggagaactgcgca 879898 SEQ ID NO: 5473 tgcgaccgtcccctttgca 1019 1038 1 6 SEQ ID NO: 5450cctcgggccatgccatggg 376 395 SEQ ID NO: 5474 cccagtgtggggccagagg 11711190 1 5 SEQ ID NO: 5451 ttgagcaaaccatatgcaa 1478 1497 SEQ ID NO: 5475ttgcagagtgtgtcttcaa 2057 2076 1 5 SEQ ID NO: 5452 cagcttcctgaggtaccaa 221 SEQ ID NO: 5476 ttggtgtctgtgatcgctg 123 142 1 4 SEQ ID NO: 5453ggtaccaaggaggaaggat 13 32 SEQ ID NO: 5477 atccagtcgactcgctacc 66 85 1 4SEQ ID NO: 5454 ctccacgactttgggatcc 51 70 SEQ ID NO: 5478ggatcgggaggagggggag 1448 1467 1 4 SEQ ID NO: 5455 caggactggtttgtcttgg108 127 SEQ ID NO: 5479 ccaagcccgactgtgcctg 2018 2037 1 4 SEQ ID NO:5456 cttctatgtcctctttccc 155 174 SEQ ID NO: 5480 gggacagacacacaagaag1076 1095 1 4 SEQ ID NO: 5457 ttctatgtcctctttccca 156 175 SEQ ID NO:5481 tgggacagacacacaagaa 1075 1094 1 4 SEQ ID NO: 5458tggttccacattcaagaga 177 196 SEQ ID NO: 5482 tctcaataatgatagacca 15491568 1 4 SEQ ID NO: 5459 tgcctctgataaaacagtt 325 344 SEQ ID NO: 5483aactctgagatcttgggca 1868 1887 1 4 SEQ ID NO: 5460 agcccggctcctgggacag1064 1083 SEQ ID NO: 5484 ctgtcctccagcctgggct 2034 2053 1 4 SEQ ID NO:5461 agtctctgacacaagtcag 1111 1130 SEQ ID NO: 5485 ctgaatggtaatggtgact1659 1678 1 4 SEQ ID NO: 5462 aaaaaggtgaatttttaaa 1237 1256 SEQ ID NO:5486 tttattaaaacgacatttt 2201 2220 1 4 SEQ ID NO: 5463acactctcaataatgatag 1545 1564 SEQ ID NO: 5487 ctatgaatgatgcctgtgt 21212140 1 4 SEQ ID NO: 5464 aaagaatgaacgtgctcca 37 56 SEQ ID NO: 5488tggacctcctgtggacttt 724 743 1 3 SEQ ID NO: 5465 ctttgggatccagtcgact 5978 SEQ ID NO: 5489 agtcagcggccgtgcaaag 1124 1143 1 3 SEQ ID NO: 5466gtgatcgctgacctcagga 132 151 SEQ ID NO: 5490 tcctctctccaaaggtcac 19111930 1 3 SEQ ID NO: 5467 ggaacgccttctatgtcct 148 167 SEQ ID NO: 5491aggactcatcactgcttcc 1748 1767 1 3 SEQ ID NO: 5468 gactgtgggcatcaatctc194 213 SEQ ID NO: 5492 gagactggaccagggagtc 357 376 1 3 SEQ ID NO: 5469ggacactgactattacagc 296 315 SEQ ID NO: 5493 gctgaacgtctgtctgtcc 518 5371 3 SEQ ID NO: 5470 aagcccccgtcccagattg 966 985 SEQ ID NO: 5494caattgtttgctggtgctt 1833 1852 1 3

TABLE 12 Selected palindromic sequences from human B-catenin Start EndStart End Source Index Index Match Index Index # B SEQ ID NO: 5495agcagcttcagtccccgcc 70 89 SEQ ID NO: 5542 ggcgacatatgcagctgct 2152 21711 5 SEQ ID NO: 5496 ccattctggtgccactacc 304 323 SEQ ID NO: 5543ggtatggaccccatgatgg 2387 2406 1 5 SEQ ID NO: 5497 tccttctctgagtggtaaa328 347 SEQ ID NO: 5544 tttattacatcaagaagga 985 1004 1 5 SEQ ID NO: 5498tctgagtggtaaaggcaat 334 353 SEQ ID NO: 5545 attgtacgtaccatgcaga 791 8101 5 SEQ ID NO: 5499 cagagggtacgagctgcta 473 492 SEQ ID NO: 5546tagctgcaggggtcctctg 2037 2056 1 5 SEQ ID NO: 5500 ctaaatgacgaggaccagg677 696 SEQ ID NO: 5547 cctgtaaatcatcctttag 2539 2558 1 5 SEQ ID NO:5501 taaatgacgaggaccaggt 678 697 SEQ ID NO: 5548 acctgtaaatcatccttta2538 2557 1 5 SEQ ID NO: 5502 gtcctgtatgagtgggaac 383 402 SEQ ID NO:5549 gttccgaatgtctgaggac 2176 2195 2 4 SEQ ID NO: 5503cccagcgccgtacgtccat 1839 1858 SEQ ID NO: 5550 atgggctgccagatctggg 24512470 2 4 SEQ ID NO: 5504 tcccctgagggtatttgaa 143 162 SEQ ID NO: 5551ttcacatcctagctcggga 1929 1948 1 4 SEQ ID NO: 5505 gggtatttgaagtatacca151 170 SEQ ID NO: 5552 tggttaagctcttacaccc 1680 1699 1 4 SEQ ID NO:5506 gctgttagtcactggcagc 260 279 SEQ ID NO: 5553 gctgcctccaggtgacagc2494 2513 1 4 SEQ ID NO: 5507 gtcctgtatgagtgggaac 383 402 SEQ ID NO:5554 gttcgccttcactatggac 1652 1671 1 4 SEQ ID NO: 5508tcctgtatgagtgggaaca 384 403 SEQ ID NO: 5555 tgttccgaatgtctgagga 21752194 1 4 SEQ ID NO: 5509 gtatgcaatgactcgagct 454 473 SEQ ID NO: 5556agctggcctggtttgatac 2517 2536 1 4 SEQ ID NO: 5510 gtccagcgtttggctgaac563 582 SEQ ID NO: 5557 gttcgccttcactatggac 1652 1671 1 4 SEQ ID NO:5511 tatcaagatgatgcagaac 623 642 SEQ ID NO: 5558 gttcgtgcacatcaggata1820 1839 1 4 SEQ ID NO: 5512 tatggtccatcagctttct 718 737 SEQ ID NO:5559 agaaagcaagctcatcata 1126 1145 1 4 SEQ ID NO: 5513ccctggtgaaaatgcttgg 915 934 SEQ ID NO: 5560 ccaaagagtagctgcaggg 20292048 1 4 SEQ ID NO: 5514 agctttaggacttcacctg 1291 1310 SEQ ID NO: 5561caggtgacagcaatcagct 2502 2521 1 4 SEQ ID NO: 5515 ggaatctttcagatgctgc1356 1375 SEQ ID NO: 5562 gcagctgctgttttgttcc 2162 2181 1 4 SEQ ID NO:5516 tgtccttcgggctggtgac 1549 1568 SEQ ID NO: 5563 gtcatctgaccagccgaca1605 1624 1 4 SEQ ID NO: 5517 cacagctcctctgacagag 2107 2126 SEQ ID NO:5564 ctctaggaatgaaggtgtg 2134 2153 1 4 SEQ ID NO: 5518ccagacagaaaagcggctg 245 264 SEQ ID NO: 5565 cagctcgttgtaccgctgg 828 8472 3 SEQ ID NO: 5519 cagcagcgttggcccggcc 4 23 SEQ ID NO: 5566ggccaccaccctggtgctg 2420 2439 1 3 SEQ ID NO: 5520 aggtctgaggagcagcttc 6079 SEQ ID NO: 5567 gaagaggatgtggatacct 359 378 1 3 SEQ ID NO: 5521actgttttgaaaatccagc 174 193 SEQ ID NO: 5568 gctgatattgatggacagt 437 4561 3 SEQ ID NO: 5522 ctgatttgatggagttgga 213 232 SEQ ID NO: 5569tccaggtgacagcaatcag 2500 2519 1 3 SEQ ID NO: 5523 ccagacagaaaagcggctg245 264 SEQ ID NO: 5570 cagcaacagtcttacctgg 275 294 1 3 SEQ ID NO: 5524acagctccttctctgagtg 323 342 SEQ ID NO: 5571 cactgagcctgccatctgt 15791598 1 3 SEQ ID NO: 5525 tggatacctcccaagtcct 369 388 SEQ ID NO: 5572aggactaaataccattcca 1972 1991 1 3 SEQ ID NO: 5526 tcaagaacaagtagctgat424 443 SEQ ID NO: 5573 atcagctggcctggtttga 2514 2533 1 3 SEQ ID NO:5527 agctcagagggtacgagct 469 488 SEQ ID NO: 5574 agctggtggaatgcaagct1276 1295 1 3 SEQ ID NO: 5528 gcatgcagatcccatctac 516 535 SEQ ID NO:5575 gtagaagctggtggaatgc 1271 1290 1 3 SEQ ID NO: 5529ccacacgtgcaatccctga 645 664 SEQ ID NO: 5576 tcagatgatataaatgtgg 14301449 1 3 SEQ ID NO: 5530 cacacgtgcaatccctgaa 646 665 SEQ ID NO: 5577ttcagatgatataaatgtg 1429 1448 1 3 SEQ ID NO: 5531 ggaccttgcataacctttc846 865 SEQ ID NO: 5578 gaaatcttgccctttgtcc 1743 1762 1 3 SEQ ID NO:5532 ctccacaaccttttattac 974 993 SEQ ID NO: 5579 gtaaatcatcctttaggag2542 2561 1 3 SEQ ID NO: 5533 cagagtgctgaaggtgcta 1222 1241 SEQ ID NO:5580 tagctgcaggggtcctctg 2037 2056 1 3 SEQ ID NO: 5534ggactctcaggaatctttc 1347 1366 SEQ ID NO: 5581 gaaatcttgccctttgtcc 17431762 1 3 SEQ ID NO: 5535 tgatataaatgtggtcacc 1435 1454 SEQ ID NO: 5582ggtgacagggaagacatca 1562 1581 1 3 SEQ ID NO: 5536 cccagcgccgtacgtccat1839 1858 SEQ ID NO: 5583 atggccaggatgccttggg 2370 2389 1 3 SEQ ID NO:5537 gtccatgggtgggacacag 1852 1871 SEQ ID NO: 5584 ctgtgaacttgctcaggac2053 2072 1 3 SEQ ID NO: 5538 ttgtaccggagcccttcac 1915 1934 SEQ ID NO:5585 gtgaacttgctcaggacaa 2055 2074 1 3 SEQ ID NO: 5539ttgttatcagaggactaaa 1962 1981 SEQ ID NO: 5586 tttaggagtaacaatacaa 25532572 1 3 SEQ ID NO: 5540 gaagctattgaagctgagg 2084 2103 SEQ ID NO: 5587cctctgacagagttacttc 2114 2133 1 3 SEQ ID NO: 5541 tcagaacagagccaatggc2247 2266 SEQ ID NO: 5588 gccaccaccctggtgctga 2421 2440 1 3

TABLE 13 Selected palindromic sequences from human hepatitis C virus(HCV) Start End Start End Source Index Index Match Index Index # B SEQID NO: 5589 cagcacctgggtgctggta 5314 5333 SEQ ID NO: 6135taccatcacccagctgctg 6196 6215 1 9 SEQ ID NO: 5590 aactcgtccggatgcccgg1882 1701 SEQ ID NO: 6136 ccgggcagcgggtcgagtt 8202 8221 1 8 SEQ ID NO:5591 cgctgctgggtagcgctca 1049 1068 SEQ ID NO: 6137 tgagagcgacgccgcagcg6151 6170 1 7 SEQ ID NO: 5592 ctccggatcccacaagccg 1352 1371 SEQ ID NO:6138 cggcatgtgggcccgggag 6053 6072 1 7 SEQ ID NO: 5593tgtaacatcgggggggtcg 2048 2067 SEQ ID NO: 6139 cgacccctcccacattaca 68716890 1 7 SEQ ID NO: 5594 gtaacatcgggggggtcgg 2049 2068 SEQ ID NO: 6140ccgacccctcccacattac 6870 6889 1 7 SEQ ID NO: 5595 cagccaccaagcaggcgga5556 5575 SEQ ID NO: 6141 tccggctggttcgttgctg 9254 9273 1 7 SEQ ID NO:5596 ctcaccacccagaacaccc 5744 5763 SEQ ID NO: 6142 gggtgtgcacggtgttgag6291 6310 1 7 SEQ ID NO: 5597 ccagccttaccatcaccca 6189 8208 SEQ ID NO:6143 tgggcgctggtatcgctgg 5832 5851 1 7 SEQ ID NO: 5598ctacgccgtgttccggctc 6249 6268 SEQ ID NO: 6144 gagcccgaaccggacgtag 68306849 1 7 SEQ ID NO: 5599 tacgccgtgttccggctcg 6250 6269 SEQ ID NO: 6145cgagcccgaaccggacgta 6829 6848 1 7 SEQ ID NO: 5600 gagttcctggtaaaagcct8216 8235 SEQ ID NO: 6146 aggctatgactaggtactc 8634 8653 1 7 SEQ ID NO:5601 atggcggggaactgggcta 1430 1449 SEQ ID NO: 6147 tagcgcattttcactccat9019 9038 2 6 SEQ ID NO: 5602 aaccaaacgtaacaccaac 370 389 SEQ ID NO:6148 gttgccgctaccttaggtt 4115 4134 1 6 SEQ ID NO: 5603ggtggtcagatcgttggtg 419 438 SEQ ID NO: 6149 caccagcccgctcaccacc 57345753 1 6 SEQ ID NO: 5604 ccttggcccctctatggca 584 603 SEQ ID NO: 6150tgccaacgtgggtacaagg 6374 6393 1 6 SEQ ID NO: 5605 taccccggccacgcgtcag1265 1284 SEQ ID NO: 6151 ctgacgactagctgcggta 8465 8484 1 6 SEQ ID NO:5606 gggcacgctgcccgcctca 1508 1527 SEQ ID NO: 6152 tgagacgacgaccgtgccc4759 4778 1 6 SEQ ID NO: 5607 ctgcaatgactccctccag 1624 1643 SEQ ID NO:6153 ctggtggccctcaatgcag 2594 2613 1 6 SEQ ID NO: 5608aaccgatcgtctcggcaac 1897 1916 SEQ ID NO: 6154 gttgccgctaccttaggtt 41154134 1 6 SEQ ID NO: 5609 gtgcggggcccccccgtgt 2032 2051 SEQ ID NO: 6155acaccacgggcccctgcac 6537 6556 1 6 SEQ ID NO: 5610 atgtggggggcgtggagca2238 2257 SEQ ID NO: 6156 tgctcaatgtcctacacat 7610 7629 1 6 SEQ ID NO:5611 ggagagcgttgcaacttgg 2288 2307 SEQ ID NO: 6157 ccaagctcaaactcactcc9207 9226 1 6 SEQ ID NO: 5612 cgtccgttgccggagcgca 2613 2632 SEQ ID NO:6158 tgcgagcccgaaccggacg 6827 6846 1 6 SEQ ID NO: 5613gtctggcattattgacctt 2817 2836 SEQ ID NO: 6159 aaggtcacctttgacagac 77637782 1 6 SEQ ID NO: 5614 tctttgatatcaccaaact 2997 3016 SEQ ID NO: 6160agttcgatgaaatggaaga 5454 5473 1 6 SEQ ID NO: 5615 cttctgattgccatactcg3014 3033 SEQ ID NO: 6161 cgagcaattcaagcagaag 5518 5537 1 6 SEQ ID NO:5616 gcggcgtgtggggacatca 3314 3333 SEQ ID NO: 6162 tgatcacgccatgcgccgc7641 7660 1 6 SEQ ID NO: 5617 gggacatcatcctgggcct 3324 3343 SEQ ID NO:6163 aggcggtggattttgtccc 3915 3934 1 6 SEQ ID NO: 5618gggcgtcttccgggccgct 3874 3893 SEQ ID NO: 6164 agcggcacggcgaccgccc 74397458 1 6 SEQ ID NO: 5619 ggcgtcttccgggccgctg 3875 3894 SEQ ID NO: 6165cagcggcacggcgaccgcc 7438 7457 1 6 SEQ ID NO: 5620 gcgtcttccgggccgctgt3876 3895 SEQ ID NO: 6166 acaggtgccctgatcacgc 7631 7650 1 6 SEQ ID NO:5621 gtccccggtcttcacagac 3961 3980 SEQ ID NO: 6167 gtcttggaagaacccggac7252 7271 1 6 SEQ ID NO: 5622 catcaggactggggtaagg 4174 4193 SEQ ID NO:6168 ccttcctcaagccgtgatg 8155 8174 1 6 SEQ ID NO: 5623ccgacggtggttgctccgg 4245 4264 SEQ ID NO: 6169 ccgggggaacggccctcgg 48534872 1 6 SEQ ID NO: 5624 ggggggaaggcacctcatt 4501 4520 SEQ ID NO: 6170aatgttgtgacttggcccc 8334 8353 1 6 SEQ ID NO: 5625 ccgagcaattcaagcagaa5517 5536 SEQ ID NO: 6171 ttctgattgccatactcgg 3015 3034 1 6 SEQ ID NO:5626 agatgaaggcaaaggcgtc 7821 7840 SEQ ID NO: 6172 gacgaccttgtcgttatct8564 8583 1 6 SEQ ID NO: 5627 cccctagggggcgctgcca 767 786 SEQ ID NO:6173 tggccggcgccccccgggg 3674 3693 3 5 SEQ ID NO: 5628ctcccggcctagttggggc 646 665 SEQ ID NO: 6174 gcccccccttgagggggag 75197538 2 5 SEQ ID NO: 5629 ttccgctcgtcggcggccc 750 769 SEQ ID NO: 6175gggcaaaggacgtccggaa 7923 7942 2 5 SEQ ID NO: 5630 cccctagggggcgctgcca767 786 SEQ ID NO: 6176 tggcgggggcccactgggg 1383 1402 2 5 SEQ ID NO:5631 gccccgccggcatgcgaca 1222 1241 SEQ ID NO: 6177 tgtcccagggggggagggc9147 9166 2 5 SEQ ID NO: 5632 aggacgaccgggtcctttc 178 197 SEQ ID NO:6178 gaaaaaggacggttgtcct 7341 7360 1 5 SEQ ID NO: 5633ggacgaccgggtcctttct 179 198 SEQ ID NO: 6179 agaaaaaggacggttgtcc 73407359 1 5 SEQ ID NO: 5634 aaaaccaaacgtaacacca 368 387 SEQ ID NO: 6180tggtttttttttttttttt 9443 9462 1 5 SEQ ID NO: 5635 caaccgccgcccacaggac385 404 SEQ ID NO: 6181 gtcctgaacccgtctgttg 4100 4119 1 5 SEQ ID NO:5636 cggtggtcagatcgttggt 418 437 SEQ ID NO: 6182 accattgagacgacgaccg4754 4773 1 5 SEQ ID NO: 5637 acctgttgccgcgcagggg 444 463 SEQ ID NO:6183 ccccggccacgcgtcaggt 1267 1286 1 5 SEQ ID NO: 5638tgccgcgcaggggccccag 450 469 SEQ ID NO: 6184 ctgggcgcgctgacgggca 31643183 1 5 SEQ ID NO: 5639 gggccccaggttgggtgtg 460 479 SEQ ID NO: 6185cacagcctgtctcgtgccc 9296 9315 1 5 SEQ ID NO: 5640 gttggggccccacggaccc657 676 SEQ ID NO: 6186 gggtgggtagccgcccaac 5783 5802 1 5 SEQ ID NO:5641 ttggggccccacggacccc 658 677 SEQ ID NO: 6187 ggggtgggtagccgcccaa5782 5801 1 5 SEQ ID NO: 5642 tggggccccacggaccccc 659 678 SEQ ID NO:6188 gggggtgggtagccgccca 5781 5800 1 5 SEQ ID NO: 5643cctcacatgcggcctcgcc 715 734 SEQ ID NO: 6189 ggcggggcgacaatagagg 37743793 1 5 SEQ ID NO: 5644 cacatgcggcctcgccgac 718 737 SEQ ID NO: 6190gtcgtcggagtcgtgtgtg 6020 6039 1 5 SEQ ID NO: 5645 tccgctcgtcggcggcccc751 770 SEQ ID NO: 6191 ggggcaaaggacgtccgga 7922 7941 1 5 SEQ ID NO:5646 ggcgctgccagggccttgg 776 795 SEQ ID NO: 6192 ccaagccacagtgtgcgcc5110 5129 1 5 SEQ ID NO: 5647 ccatgtcacgaacgactgc 943 962 SEQ ID NO:6193 gcagcaacacgtggcatgg 6498 6517 1 5 SEQ ID NO: 5648gtgccctgcgttcgggagg 1019 1038 SEQ ID NO: 6194 cctcacaacgggggggcac 14951514 1 5 SEQ ID NO: 5649 tgccctgcgttcgggaggg 1020 1039 SEQ ID NO: 6195ccctcacaacgggggggca 1494 1513 1 5 SEQ ID NO: 5650 gccctgcgttcgggagggt1021 1040 SEQ ID NO: 6196 accctcacaacgggggggc 1493 1512 1 5 SEQ ID NO:5651 aggaatgctaccatcccca 1085 1104 SEQ ID NO: 6197 tgggcatcggcacagtcct4323 4342 1 5 SEQ ID NO: 5652 tccccactacgacaatacg 1098 1117 SEQ ID NO:6198 cgtattcccagatttggga 8092 8111 1 5 SEQ ID NO: 5653atacgacaccacgtcgatt 1112 1131 SEQ ID NO: 6199 aatcaatgctgtagcgtat 45764595 1 5 SEQ ID NO: 5654 atttgctcgttggggcggc 1128 1147 SEQ ID NO: 6200gccgccacttgcggcaaat 9164 9183 1 5 SEQ ID NO: 5655 ccttctcgccccgccggca1215 1234 SEQ ID NO: 6201 tgccaacgtgggtacaagg 6374 6393 1 5 SEQ ID NO:5656 accccggccacgcgtcagg 1266 1285 SEQ ID NO: 6202 cctgccgcggttaccgggt6340 6359 1 5 SEQ ID NO: 5657 gccctcgtagtgtcgcagt 1331 1350 SEQ ID NO:6203 actgcgtcggcatgtgggc 6046 6065 1 5 SEQ ID NO: 5658gccgtctcagagaatccag 1558 1577 SEQ ID NO: 6204 ctggtatcgctggtgcggc 58385857 1 5 SEQ ID NO: 5659 ctgaactgcaatgactccc 1619 1638 SEQ ID NO: 6205gggacagatcggagctcag 2313 2332 1 5 SEQ ID NO: 5660 agactgggtttcttgccgc1641 1660 SEQ ID NO: 6206 gcggcgagcctacgagtct 8609 8628 1 5 SEQ ID NO:5661 tcgtccggatgcccggagc 1685 1704 SEQ ID NO: 6207 gctccgggggcgcttacga4257 4276 1 5 SEQ ID NO: 5662 ccagggatggggtcctatc 1738 1757 SEQ ID NO:6208 gataacttcccctacctgg 5084 5103 1 5 SEQ ID NO: 5663gacaaccgatcgtctcggc 1894 1913 SEQ ID NO: 6209 gccgcggttaccgggtgtc 63436362 1 5 SEQ ID NO: 5664 caagacgtgcggggccccc 2026 2045 SEQ ID NO: 6210ggggtctcccccctccttg 6919 6938 1 5 SEQ ID NO: 5665 acgtgcggggcccccccgt2030 2049 SEQ ID NO: 6211 acgggcgcccccattacgt 4202 4221 1 5 SEQ ID NO:5666 ccggaagcaccccgaggcc 2101 2120 SEQ ID NO: 6212 ggccgctgtatgcacccgg3886 3905 1 5 SEQ ID NO: 5667 aggccacgtactcaaaatg 2115 2134 SEQ ID NO:6213 cattatgtccaaatggcct 3137 3156 1 5 SEQ ID NO: 5668tgtatgtggggggcgtgga 2235 2254 SEQ ID NO: 6214 tccaagtggcccatctaca 40114030 1 5 SEQ ID NO: 5669 gagtggcaggttctgccct 2354 2373 SEQ ID NO: 6215agggcaggggtggcgactc 3400 3419 1 5 SEQ ID NO: 5670 tcctttgcaatcaaatggg2474 2493 SEQ ID NO: 6216 cccaccttatgggcaagga 8861 8880 1 5 SEQ ID NO:5671 agcccaggccgaggccgcc 2566 2585 SEQ ID NO: 6217 ggcgtccacagtcaaggct7834 7853 1 5 SEQ ID NO: 5672 ggcggcatatgctttctat 2698 2717 SEQ ID NO:6218 atagaagaagcctgccgcc 7865 7884 1 5 SEQ ID NO: 5673gcggcatatgctttctatg 2699 2718 SEQ ID NO: 6219 catagaagaagcctgccgc 78647883 1 5 SEQ ID NO: 5674 cggcatatgctttctatgg 2700 2719 SEQ ID NO: 6220ccatagaagaagcctgccg 7863 7882 1 5 SEQ ID NO: 5675 tgcatgtgtgggttccccc2913 2932 SEQ ID NO: 6221 ggggggacggcatcatgca 6402 6421 1 5 SEQ ID NO:5676 cccccctcaacgtccgggg 2928 2947 SEQ ID NO: 6222 ccccaatcgatgaacgggg9376 9395 1 5 SEQ ID NO: 5677 gggcaggggtggcgactcc 3401 3420 SEQ ID NO:6223 ggaggccgcaagccagccc 8068 8085 1 5 SEQ ID NO: 5678atgttggactgtctaccat 3574 3593 SEQ ID NO: 6224 atggtaccgaccctaacat 41584177 1 5 SEQ ID NO: 5679 tgttggactgtctaccatg 3575 3594 SEQ ID NO: 6225catggtaccgaccctaaca 4157 4176 1 5 SEQ ID NO: 5680 cgttccctgacaccatgca3695 3714 SEQ ID NO: 6226 tgcacgatgctcgtgaacg 8543 8562 1 5 SEQ ID NO:5681 acaccatgcacctgtggca 3704 3723 SEQ ID NO: 6227 tgccgcggttaccgggtgt6342 6361 1 5 SEQ ID NO: 5682 caccatgcacctgtggcag 3705 3724 SEQ ID NO:6228 ctgccgcggttaccgggtg 6341 6360 1 5 SEQ ID NO: 5683ggcatcggcacagtcctgg 4325 4344 SEQ ID NO: 6229 ccaggattgcccgtttgcc 49794998 1 5 SEQ ID NO: 5684 aagcggagacggctggagc 4347 4366 SEQ ID NO: 6230gctccccccagcgctgctt 5804 5823 1 5 SEQ ID NO: 5685 ggagcgcggcttgtcgtgc4361 4380 SEQ ID NO: 6231 gcacggcgaccgcccctcc 7443 7462 1 5 SEQ ID NO:5686 cgaagccatcaagggggga 4489 4508 SEQ ID NO: 6232 tccccccagcgctgcttcg5806 5825 1 5 SEQ ID NO: 5687 tggaagtgtctcatacggc 5165 5184 SEQ ID NO:6233 gccggattacaatcctcca 7225 7244 1 5 SEQ ID NO: 5688gggtgctggtaggcggagt 5322 5341 SEQ ID NO: 6234 actcgcgatcccaccaccc 87658784 1 5 SEQ ID NO: 5689 gtgggtaggatcatcttgt 5390 5409 SEQ ID NO: 6235acaacatggtctacgccac 7713 7732 1 5 SEQ ID NO: 5690 cgccgagcaattcaagcag5515 5534 SEQ ID NO: 6236 ctgcacgccttccccggcg 6550 6569 1 5 SEQ ID NO:5691 tggagtccaagtggcgagc 5592 5611 SEQ ID NO: 6237 gctcctcatacggattcca8175 8194 1 5 SEQ ID NO: 5692 tggcgagctttggagacct 5603 5622 SEQ ID NO:6238 aggtgccctgatcacgcca 7633 7652 1 5 SEQ ID NO: 5693gcccgctcaccacccagaa 5739 5758 SEQ ID NO: 6239 ttctggcgggctatggggc 58955914 1 5 SEQ ID NO: 5694 tgagtgacttcaagacctg 6306 6325 SEQ ID NO: 6240caggctataaaatcgctca 8363 8382 1 5 SEQ ID NO: 5695 atgtcaaaaacggttccat6456 6475 SEQ ID NO: 6241 atggtaccgaccctaacat 4158 4177 1 5 SEQ ID NO:5696 ccgaaaacctgcagcaaca 6488 6507 SEQ ID NO: 6242 tgttcctccaatgtgtcgg8708 8727 1 5 SEQ ID NO: 5697 ggcgccaaactattccaag 6565 6584 SEQ ID NO:6243 cttgaaagcctctgccgcc 8500 8519 1 5 SEQ ID NO: 5698gccctccttgagggcgaca 6967 6986 SEQ ID NO: 6244 tgtctcctacttgaagggc 38143833 1 5 SEQ ID NO: 5699 cacccgcgtggagtcggag 7078 7097 SEQ ID NO: 6245ctccggtggtacacgggtg 7278 7297 1 5 SEQ ID NO: 5700 ggagggggatgagaatgaa7138 7157 SEQ ID NO: 6246 ttcatgctgtgcctactcc 9326 9345 1 5 SEQ ID NO:5701 gcggcgatacccatatggg 7202 7221 SEQ ID NO: 6247 cccagggggggagggccgc9150 9169 1 5 SEQ ID NO: 5702 ttgccacctgtcaaggccc 7301 7320 SEQ ID NO:6248 gggccgccacttgcggcaa 9162 9181 1 5 SEQ ID NO: 5703cccccccttgagggggagc 7520 7539 SEQ ID NO: 6249 gctcccggcctagttgggg 645664 1 5 SEQ ID NO: 5704 ctgctgctcaatgtcctac 7606 7625 SEQ ID NO: 6250gtaggactggcaggggcag 4809 4828 1 5 SEQ ID NO: 5705 catggacaggtgccctgat7626 7645 SEQ ID NO: 6251 atcattgaacgactccatg 8996 9015 1 5 SEQ ID NO:5706 atggacaggtgccctgatc 7627 7646 SEQ ID NO: 6252 gatcattgaacgactccat8995 9014 1 5 SEQ ID NO: 5707 ggctatgactaggtactcc 8635 8654 SEQ ID NO:6253 ggagcaacttgaaaaagcc 8920 8939 1 5 SEQ ID NO: 5708caccatagatcactcccct 27 46 SEQ ID NO: 6254 agggccttggcacatggtg 785 804 24 SEQ ID NO: 5709 agctgttcaccttctcgcc 1206 1225 SEQ ID NO: 6255ggcgtgctgacgactagct 8459 8478 2 4 SEQ ID NO: 5710 ctgcaatgactccctccag1624 1643 SEQ ID NO: 6256 ctggtgcggctgttggcag 5847 5866 2 4 SEQ ID NO:5711 atgtggggggcgtggagca 2238 2257 SEQ ID NO: 6257 tgctgcgccatcacaacat7701 7720 2 4 SEQ ID NO: 5712 tggggacatcatcctgggc 3322 3341 SEQ ID NO:6258 gcccaactcgctcccccca 5795 5814 2 4 SEQ ID NO: 5713gggacatcatcctgggcct 3324 3343 SEQ ID NO: 6259 aggcaggagataacttccc 50765095 2 4 SEQ ID NO: 5714 gggagatactcctggggcc 3366 3385 SEQ ID NO: 6260ggcccctgcacgccttccc 6545 6564 2 4 SEQ ID NO: 5715 atgttggactgtctaccat3574 3593 SEQ ID NO: 6261 atggtctacgccacgacat 7718 7737 2 4 SEQ ID NO:5716 ccagccttaccatcaccca 6189 6208 SEQ ID NO: 6262 tgggtacaagggagtctgg6382 6401 2 4 SEQ ID NO: 5717 gccctccttgagggcgaca 6967 6986 SEQ ID NO:6263 tgtcccagggggggagggc 9147 9166 2 4 SEQ ID NO: 5718ccagcccccgattgggggc 1 20 SEQ ID NO: 6264 gcccgagggcagggcctgg 550 569 1 4SEQ ID NO: 5719 accatagatcactcccctg 28 47 SEQ ID NO: 6265cagggccttggcacatggt 784 803 1 4 SEQ ID NO: 5720 atgagtgtcgtgcagcctc 95114 SEQ ID NO: 6266 gaggccgcgatgccatcat 2946 2965 1 4 SEQ ID NO: 5721gtgcagcctccaggacccc 104 123 SEQ ID NO: 6267 gggggacggcatcatgcac 64036422 1 4 SEQ ID NO: 5722 tgcagcctccaggaccccc 105 124 SEQ ID NO: 6268ggggggacggcatcatgca 6402 6421 1 4 SEQ ID NO: 5723 ccaggaccccccctcccgg113 132 SEQ ID NO: 6269 ccggctggttcgttgctgg 9255 9274 1 4 SEQ ID NO:5724 accccccctcccgggagag 118 137 SEQ ID NO: 6270 ctctcatgccaacgtgggt6368 6387 1 4 SEQ ID NO: 5725 ccccctcccgggagagcca 121 140 SEQ ID NO:6271 tggcaatgagggcatgggg 598 617 1 4 SEQ ID NO: 5726 agactgctagccgagtagt243 262 SEQ ID NO: 6272 actatgcggtccccggtct 3953 3972 1 4 SEQ ID NO:5727 agccgagtagtgttgggtc 251 270 SEQ ID NO: 6273 gaccaggatctcgtcggct3656 3675 1 4 SEQ ID NO: 5728 ggtgcttgcgagtgccccg 299 318 SEQ ID NO:6274 cggggccttggttgacacc 2139 2158 1 4 SEQ ID NO: 5729gcgagtgccccgggaggtc 306 325 SEQ ID NO: 6275 gacccccggcgtaggtcgc 671 6901 4 SEQ ID NO: 5730 accgtgcaccatgagcacg 331 350 SEQ ID NO: 6276cgtgcaatacctgtacggt 2437 2456 1 4 SEQ ID NO: 5731 cccgggcggtggtcagatc412 431 SEQ ID NO: 6277 gatcatgcatactcccggg 997 1016 1 4 SEQ ID NO: 5732gccgcgcaggggccccagg 451 470 SEQ ID NO: 6278 cctgcacgccttccccggc 65496568 1 4 SEQ ID NO: 5733 accccgtggaaggcgacag 511 530 SEQ ID NO: 6279ctgtatgcacccggggggt 3891 3910 1 4 SEQ ID NO: 5734 ccccgtggaaggcgacagc512 531 SEQ ID NO: 6280 gctgtatgcacccgggggg 3890 3909 1 4 SEQ ID NO:5735 agcctatccccaaggctcg 528 547 SEQ ID NO: 6281 cgagggcagggcctgggct 553572 1 4 SEQ ID NO: 5736 ctatccccaaggctcgccg 531 550 SEQ ID NO: 6282cggctgtcgttcccgatag 5418 5437 1 4 SEQ ID NO: 5737 tatccccaaggctcgccgg532 551 SEQ ID NO: 6283 ccggctgtcgttcccgata 5417 5436 1 4 SEQ ID NO:5738 cgggtatccttggcccctc 577 596 SEQ ID NO: 6284 gaggccgcaagccagcccg8067 8086 1 4 SEQ ID NO: 5739 gcatggggtgggcaggatg 609 628 SEQ ID NO:6285 catcgataccctcacatgc 706 725 1 4 SEQ ID NO: 5740 tcctgtcaccccgcggctc630 649 SEQ ID NO: 6286 gagctgcaaagctccagga 8523 8542 1 4 SEQ ID NO:5741 gggccccacggacccccgg 661 680 SEQ ID NO: 6287 ccggccgcatatgcggccc4064 4083 1 4 SEQ ID NO: 5742 ggccccacggacccccggc 662 681 SEQ ID NO:6288 gccggccgcatatgcggcc 4063 4082 1 4 SEQ ID NO: 5743cggcctcgccgacctcatg 724 743 SEQ ID NO: 6289 catgaggatcatcgggccg 64726491 1 4 SEQ ID NO: 5744 ggcctcgccgacctcatgg 725 744 SEQ ID NO: 6290ccatgaggatcatcgggcc 6471 6490 1 4 SEQ ID NO: 5745 ggccccctagggggcgctg764 783 SEQ ID NO: 6291 cagctccgaattgtcggcc 7414 7433 1 4 SEQ ID NO:5746 tggcacatggtgtccgggt 792 811 SEQ ID NO: 6292 acccacgctgcacgggcca5188 5207 1 4 SEQ ID NO: 5747 cttcctcttggctctgctg 868 887 SEQ ID NO:6293 cagcataggtcttgggaag 5863 5882 1 4 SEQ ID NO: 5748catgtcacgaacgactgct 944 963 SEQ ID NO: 6294 agcagtgctcacttccatg 68476866 1 4 SEQ ID NO: 5749 gaggcggcggacttgatca 983 1002 SEQ ID NO: 6295tgatggcattcacagcctc 5712 5731 1 4 SEQ ID NO: 5750 catccccactacgacaata1096 1115 SEQ ID NO: 6296 tattaccggggtcttgatg 4592 4611 1 4 SEQ ID NO:5751 gctgttcaccttctcgccc 1207 1226 SEQ ID NO: 6297 gggctgcgtgggaaacagc8793 8812 1 4 SEQ ID NO: 5752 gccccgccggcatgcgaca 1222 1241 SEQ ID NO:6298 tgtctcctacttgaagggc 3814 3833 1 4 SEQ ID NO: 5753tggcctgggacatgatgat 1293 1312 SEQ ID NO: 6299 atcaatttgctccctgcca 59816000 1 4 SEQ ID NO: 5754 cacaagccgtcatcgacat 1362 1381 SEQ ID NO: 6300atgtttgggactgggtgtg 6279 6298 1 4 SEQ ID NO: 5755 agccgtcatcgacatggtg1366 1385 SEQ ID NO: 6301 caccaagcaggcggaggct 5560 5579 1 4 SEQ ID NO:5756 ggtggcgggggcccactgg 1381 1400 SEQ ID NO: 6302 ccagggctcaggccccacc5127 5146 1 4 SEQ ID NO: 5757 gggggcccactggggagtc 1387 1406 SEQ ID NO:6303 gactaggtactccgccccc 8641 8660 1 4 SEQ ID NO: 5758atggcggggaactgggcta 1430 1449 SEQ ID NO: 6304 tagcagtgctcacttccat 68466865 1 4 SEQ ID NO: 5759 ttgattgtgatgctacttt 1454 1473 SEQ ID NO: 6305aaagcaagctgcccatcaa 7665 7684 1 4 SEQ ID NO: 5760 caacgggggggcacgctgc1500 1519 SEQ ID NO: 6306 gcagaaggcgctcgggttg 5530 5549 1 4 SEQ ID NO:5761 acgctgcccgcctcaccag 1512 1531 SEQ ID NO: 6307 ctggacccgaggagagcgt2278 2297 1 4 SEQ ID NO: 5762 tcagagaatccagcttata 1564 1583 SEQ ID NO:6308 tatatcgggggtcccctga 8393 8412 1 4 SEQ ID NO: 5763accaatggcagttggcaca 1586 1605 SEQ ID NO: 6309 tgtggctcggggccttggt 21322151 1 4 SEQ ID NO: 5764 ccaatggcagttggcacat 1587 1606 SEQ ID NO: 6310atgtggctcggggccttgg 2131 2150 1 4 SEQ ID NO: 5765 gtcctatcacttatgctga1749 1768 SEQ ID NO: 6311 tcaggactggggtaaggac 4176 4195 1 4 SEQ ID NO:5766 ctgagcctacaaaagaccc 1764 1783 SEQ ID NO: 6312 gggtggcttcatgcctcag9063 9082 1 4 SEQ ID NO: 5767 caggtgtgtggtccagtgt 1844 1863 SEQ ID NO:6313 acactccagttaactcctg 8817 8836 1 4 SEQ ID NO: 5768tgtggtccagtgtattgct 1850 1869 SEQ ID NO: 6314 agcagggccatcaaccaca 79497968 1 4 SEQ ID NO: 5769 gcttcaccccaagtcctgt 1866 1885 SEQ ID NO: 6315acagcagaggcggctaagc 6887 6906 1 4 SEQ ID NO: 5770 ctgttgtcgtggggacaac1881 1900 SEQ ID NO: 6316 gttgcaacttggacgacag 2295 2314 1 4 SEQ ID NO:5771 gccgccgcaaggcaactgg 1972 1991 SEQ ID NO: 6317 ccagttggacttatccggc9241 9260 1 4 SEQ ID NO: 5772 ggcaactggttcggctgta 1982 2001 SEQ ID NO:6318 tacacgggtgcccattgcc 7287 7306 1 4 SEQ ID NO: 5773gcaactggttcggctgtac 1983 2002 SEQ ID NO: 6319 gtacacgggtgcccattgc 72867305 1 4 SEQ ID NO: 5774 ccccgtgtaacatcggggg 2043 2062 SEQ ID NO: 6320ccccaatcgatgaacgggg 9376 9395 1 4 SEQ ID NO: 5775 ggactgcttccggaagcac2092 2111 SEQ ID NO: 6321 gtgctggtaggcggagtcc 5324 5343 1 4 SEQ ID NO:5776 gactgcttccggaagcacc 2093 2112 SEQ ID NO: 6322 ggtgctggtaggcggagtc5323 5342 1 4 SEQ ID NO: 5777 tccggaagcaccccgaggc 2100 2119 SEQ ID NO:6323 gcctacgagtcttcacgga 8616 8635 1 4 SEQ ID NO: 5778actcaaaatgtggctcggg 2124 2143 SEQ ID NO: 6324 cccgggcagcgggtcgagt 82018220 1 4 SEQ ID NO: 5779 ggccttggttgacacctag 2142 2161 SEQ ID NO: 6325ctagccggcccaaaaggcc 3611 3630 1 4 SEQ ID NO: 5780 aggagagcgttgcaacttg2287 2306 SEQ ID NO: 6326 caagccgtgatgggctcct 8162 8181 1 4 SEQ ID NO:5781 ggacagatcggagctcagc 2314 2333 SEQ ID NO: 6327 gctgggggtcattatgtcc3128 3147 1 4 SEQ ID NO: 5782 cagatcggagctcagcccg 2317 2336 SEQ ID NO:6328 cgggtggcccactgctctg 3837 3856 1 4 SEQ ID NO: 5783ggagctcagcccgctgctg 2323 2342 SEQ ID NO: 6329 cagctgctgaagaggctcc 62066225 1 4 SEQ ID NO: 5784 caccctaccggctctgtcc 2383 2402 SEQ ID NO: 6330ggactgggtgtgcacggtg 6286 6305 1 4 SEQ ID NO: 5785 cggctctgtccactggctt2391 2410 SEQ ID NO: 6331 aagcaggcggaggctgccg 5564 5583 1 4 SEQ ID NO:5786 ccatcagaacatcgtggac 2419 2438 SEQ ID NO: 6332 gtccccgttgagtccatgg3929 3948 1 4 SEQ ID NO: 5787 ggtcagcggttgtctcctt 2460 2479 SEQ ID NO:6333 aaggatgattctgatgacc 8875 8894 1 4 SEQ ID NO: 5788gccgccttagagaacctgg 2579 2598 SEQ ID NO: 6334 ccagttggacttatccggc 92419260 1 4 SEQ ID NO: 5789 gccttagagaacctggtgg 2582 2601 SEQ ID NO: 6335ccaccaagcaggcggaggc 5559 5578 1 4 SEQ ID NO: 5790 gccggagcgcacggcatcc2621 2640 SEQ ID NO: 6336 ggattgggcccacgccggc 3214 3233 1 4 SEQ ID NO:5791 gctgcatcgtgcggaggcg 2786 2805 SEQ ID NO: 6337 cgccacgacatcccgcagc7726 7745 1 4 SEQ ID NO: 5792 attattgaccttgtcgcca 2824 2843 SEQ ID NO:6338 tggcaacagacgctctaat 4647 4666 1 4 SEQ ID NO: 5793tcgccatattacaaggtgt 2837 2856 SEQ ID NO: 6339 acacaatctttcctggcga 35393558 1 4 SEQ ID NO: 5794 cgccatattacaaggtgtt 2838 2857 SEQ ID NO: 6340aacacaatctttcctggcg 3538 3557 1 4 SEQ ID NO: 5795 gtccggggaggccgcgatg2939 2958 SEQ ID NO: 6341 catcggcacagtcctggac 4327 4346 1 4 SEQ ID NO:5796 tcaccccactgcgggattg 3201 3220 SEQ ID NO: 6342 caatttaccaatgttgtga8325 8344 1 4 SEQ ID NO: 5797 ttgggcccacgccggccta 3217 3236 SEQ ID NO:6343 taggctaggggccgtccaa 5221 5240 1 4 SEQ ID NO: 5798ctacgggaccttgcggtag 3233 3252 SEQ ID NO: 6344 ctactcctactttctgtag 93389357 1 4 SEQ ID NO: 5799 cctgtcgtcttctctgaca 3260 3279 SEQ ID NO: 6345tgtcctacacatggacagg 7617 7636 1 4 SEQ ID NO: 5800 ctgtcgtcttctctgacat3261 3280 SEQ ID NO: 6346 atgtcctacacatggacag 7616 7635 1 4 SEQ ID NO:5801 cctggggggcagacaccgc 3297 3316 SEQ ID NO: 6347 gcggggtaggactggcagg4804 4823 1 4 SEQ ID NO: 5802 gggggcagacaccgcggcg 3301 3320 SEQ ID NO:6348 cgcccaactcgctcccccc 5794 5813 1 4 SEQ ID NO: 5803ggcgtgtggggacatcatc 3316 3335 SEQ ID NO: 6349 gatgttattccggtgcgcc 37553774 1 4 SEQ ID NO: 5804 tggggccggccgatagtct 3378 3397 SEQ ID NO: 6350agacgacgaccgtgcccca 4761 4780 1 4 SEQ ID NO: 5805 gaaccaggtcgagggggag3499 3518 SEQ ID NO: 6351 ctccacctatggcaagttc 4222 4241 1 4 SEQ ID NO:5806 gagggggaggttcaagtgg 3509 3528 SEQ ID NO: 6352 ccacctgtcaaggcccctc7304 7323 1 4 SEQ ID NO: 5807 aggcccaatcgcccagatg 3625 3644 SEQ ID NO:6353 catcccgcagcgcgggcct 7734 7753 1 4 SEQ ID NO: 5808ggcccaatcgcccagatgt 3626 3645 SEQ ID NO: 6354 acatcccgcagcgcgggcc 77337752 1 4 SEQ ID NO: 5809 caggatctcgtcggctggc 3659 3678 SEQ ID NO: 6355gccaataggccatttcctg 9410 9429 1 4 SEQ ID NO: 5810 aggatctcgtcggctggcc3660 3679 SEQ ID NO: 6356 ggccaataggccatttcct 9409 9428 1 4 SEQ ID NO:5811 gccccccggggcgcgttcc 3682 3701 SEQ ID NO: 6357 ggaacctatccagcagggc7938 7957 1 4 SEQ ID NO: 5812 gcacctgtggcagctcgga 3711 3730 SEQ ID NO:6358 tccggtggtacacgggtgc 7279 7298 1 4 SEQ ID NO: 5813ctgtggcagctcggacctt 3715 3734 SEQ ID NO: 6359 aaggcaaaggcgtccacag 78267845 1 4 SEQ ID NO: 5814 gcggggcgacaatagaggg 3775 3794 SEQ ID NO: 6360ccctgcctgggaaccccgc 5682 5701 1 4 SEQ ID NO: 5815 ggagcttgctctcccccag3792 3811 SEQ ID NO: 6361 ctggttgggtcacagctcc 6806 6825 1 4 SEQ ID NO:5816 gagcttgctctcccccagg 3793 3812 SEQ ID NO: 6362 cctggttgggtcacagctc6805 6824 1 4 SEQ ID NO: 5817 acttgaagggctcttcggg 3822 3841 SEQ ID NO:6363 cccgtggtggagtccaagt 5585 5604 1 4 SEQ ID NO: 5818tgtccccgttgagtccatg 3928 3947 SEQ ID NO: 6364 catggtctacgccacgaca 77177736 1 4 SEQ ID NO: 5819 gaaactactatgcggtccc 3947 3966 SEQ ID NO: 6365gggaaggcacctcattttc 4504 4523 1 4 SEQ ID NO: 5820 aaactactatgcggtcccc3948 3967 SEQ ID NO: 6366 ggggggcatatacaggttt 4828 4847 1 4 SEQ ID NO:5821 ctcccactggcagcggcaa 4032 4051 SEQ ID NO: 6367 ttgccaggaccatctggag4993 5012 1 4 SEQ ID NO: 5822 ggcgtatatgtctaaagca 4138 4157 SEQ ID NO:6368 tgctcgccaccgctacgcc 4377 4396 1 4 SEQ ID NO: 5823gcgtatatgtctaaagcac 4139 4158 SEQ ID NO: 6369 gtgctcgccaccgctacgc 43764395 1 4 SEQ ID NO: 5824 tggggtaaggaccattacc 4183 4202 SEQ ID NO: 6370ggtaaccatgtctccccca 6119 6138 1 4 SEQ ID NO: 5825 accattaccacgggcgccc4193 4212 SEQ ID NO: 6371 gggcgctggtatcgctggt 5833 5852 1 4 SEQ ID NO:5826 cgtactccacctatggcaa 4218 4237 SEQ ID NO: 6372 ttgccccaaccagaatacg8669 8688 1 4 SEQ ID NO: 5827 cagtcctggaccaagcgga 4335 4354 SEQ ID NO:6373 tccgtgagccgcatgactg 9560 9579 1 4 SEQ ID NO: 5828aggggggaaggcacctcat 4500 4519 SEQ ID NO: 6374 atgagcggcgaggcgccct 59485967 1 4 SEQ ID NO: 5829 cactccaagaagaagtgcg 4526 4545 SEQ ID NO: 6375cgcatgactgcagagagtg 9569 9588 1 4 SEQ ID NO: 5830 atcaatgctgtagcgtatt4577 4596 SEQ ID NO: 6376 aatacgacttggagttgat 8682 8701 1 4 SEQ ID NO:5831 cataccgaccagcggagac 4618 4637 SEQ ID NO: 6377 gtctcccccacgcactatg6128 6147 1 4 SEQ ID NO: 5832 aggactggcaggggcaggg 4811 4830 SEQ ID NO:6378 ccctgccatcctctctcct 5992 6011 1 4 SEQ ID NO: 5833gggaacggccctcgggcat 4857 4876 SEQ ID NO: 6379 atgctcaccgacccctccc 68636882 1 4 SEQ ID NO: 5834 cgggcatgttcgattcctc 4869 4888 SEQ ID NO: 6380gaggccgcaagccagcccg 8067 8086 1 4 SEQ ID NO: 5835 tggtacgagctcacccccg4922 4941 SEQ ID NO: 6381 cggggacttgccccaacca 8662 8681 1 4 SEQ ID NO:5836 gggcttacctaaatacacc 4962 4981 SEQ ID NO: 6382 ggtggctccatcttagccc9518 9537 1 4 SEQ ID NO: 5837 ggcttacctaaatacacca 4963 4982 SEQ ID NO:6383 tggtggctccatcttagcc 9517 9536 1 4 SEQ ID NO: 5838gagataacttcccctacct 5082 5101 SEQ ID NO: 6384 aggttggccagggggtctc 69086927 1 4 SEQ ID NO: 5839 cccacctccatcgtgggat 5140 5159 SEQ ID NO: 6385atccaagtttggctatggg 7906 7925 1 4 SEQ ID NO: 5840 catggcatgcatgtcggcc5278 5297 SEQ ID NO: 6386 ggcctctctgcagatcatg 9596 9615 1 4 SEQ ID NO:5841 ggccgacctggaagtcgtc 5293 5312 SEQ ID NO: 6387 gacgcccccacattcggcc7885 7904 1 4 SEQ ID NO: 5842 gccgacctggaagtcgtca 5294 5313 SEQ ID NO:6388 tgacgcccccacattcggc 7884 7903 1 4 SEQ ID NO: 5843tggaagtcgtcaccagcac 5301 5320 SEQ ID NO: 6389 gtgcccatgtcaggttcca 66766695 1 4 SEQ ID NO: 5844 gcacctgggtgctggtagg 5316 5335 SEQ ID NO: 6390cctacacatggacaggtgc 7620 7639 1 4 SEQ ID NO: 5845 ggttatcgtgggtaggatc5383 5402 SEQ ID NO: 6391 gatcatcgggccgaaaacc 6478 6497 1 4 SEQ ID NO:5846 cccgatagggaagtcctct 5429 5448 SEQ ID NO: 6392 agagcggctttatatcggg8383 8402 1 4 SEQ ID NO: 5847 tgaaatggaagaatgcgcc 5461 5480 SEQ ID NO:6393 ggcgcgctcgtggccttca 5924 5943 1 4 SEQ ID NO: 5848ccaagtggcgagctttgga 5598 5617 SEQ ID NO: 6394 tccattgttagagtcttgg 72407259 1 4 SEQ ID NO: 5849 ttcatcagcgggatacagt 5645 5664 SEQ ID NO: 6395actgcacgatgctcgtgaa 8541 8560 1 4 SEQ ID NO: 5850 agcgggcttatccaccctg5668 5687 SEQ ID NO: 6396 caggggtggctggcgcgct 5913 5932 1 4 SEQ ID NO:5851 ccagcccgctcaccaccca 5736 5755 SEQ ID NO: 6397 tgggcgctggtatcgctgg5832 5851 1 4 SEQ ID NO: 5852 gtgggcgctggtatcgctg 5831 5850 SEQ ID NO:6398 cagcagggccatcaaccac 7948 7967 1 4 SEQ ID NO: 5853ggaaggtgctagtggacat 5877 5896 SEQ ID NO: 6399 atgtggtctccacccttcc 81428161 1 4 SEQ ID NO: 5854 ggtcatgagcggcgaggcg 5944 5963 SEQ ID NO: 6400cgcccctcctgaccagacc 7453 7472 1 4 SEQ ID NO: 5855 catgtgggcccgggagagg6056 6075 SEQ ID NO: 6401 cctccttgagggcgacatg 6969 6988 1 4 SEQ ID NO:5856 atgtgggcccgggagaggg 6057 6076 SEQ ID NO: 6402 ccctccttgagggcgacat6968 6987 1 4 SEQ ID NO: 5857 ggggccgtgcagtggatga 6074 6093 SEQ ID NO:6403 tcatgctcctctatgcccc 7505 7524 1 4 SEQ ID NO: 5858gcgttcgcttcgcggggta 6104 6123 SEQ ID NO: 6404 taccaccacgagcttacgc 27512770 1 4 SEQ ID NO: 5859 ggggtaaccatgtctcccc 6117 6136 SEQ ID NO: 6405gggggagccgggggacccc 7531 7550 1 4 SEQ ID NO: 5860 catcacccagctgctgaag6199 6218 SEQ ID NO: 6406 cttcgagcggagggggatg 7130 7149 1 4 SEQ ID NO:5861 aggactgttctacgccgtg 6240 6259 SEQ ID NO: 6407 cacggcgaccgcccctcct7444 7463 1 4 SEQ ID NO: 5862 ttcaagacctggctccagt 6314 6333 SEQ ID NO:6408 actgcacgatgctcgtgaa 8541 8560 1 4 SEQ ID NO: 5863ctcctgccgcggttaccgg 6338 6357 SEQ ID NO: 6409 ccgggacgtgcttaaggag 78047823 1 4 SEQ ID NO: 5864 caccacgggcccctgcacg 6538 6557 SEQ ID NO: 6410cgtggaggtcacgcgggtg 6613 6632 1 4 SEQ ID NO: 5865 ggaggtcacgcgggtgggg6616 6635 SEQ ID NO: 6411 cccctccaataccacctcc 7317 7336 1 4 SEQ ID NO:5866 gaggtcacgcgggtggggg 6617 6636 SEQ ID NO: 6412 cccctcctgaccagacctc7455 7474 1 4 SEQ ID NO: 5867 atgtcaggttccagctcct 6682 6701 SEQ ID NO:6413 aggagatgggcggaaacat 7059 7078 1 4 SEQ ID NO: 5868atgaaatatccattgcggc 7152 7171 SEQ ID NO: 6414 gccgtgatgggctcctcat 81658184 1 4 SEQ ID NO: 5869 ctccattgttagagtcttg 7239 7258 SEQ ID NO: 6415caagtggcgagctttggag 5599 5618 1 4 SEQ ID NO: 5870 tgcccattgccacctgtca7295 7314 SEQ ID NO: 6416 tgactaattcaaaagggca 8409 8428 1 4 SEQ ID NO:5871 accacctccacggagaaaa 7327 7346 SEQ ID NO: 6417 ttttttccctctttatggt9502 9521 1 4 SEQ ID NO: 5872 ccacctccacggagaaaaa 7328 7347 SEQ ID NO:6418 ttttccctctttatggtgg 9504 9523 1 4 SEQ ID NO: 5873acctccacggagaaaaagg 7330 7349 SEQ ID NO: 6419 cctttgacagactgcaggt 77707789 1 4 SEQ ID NO: 5874 ggttgtcctgacggactcc 7351 7370 SEQ ID NO: 6420ggagctcgctaccaaaacc 7390 7409 1 4 SEQ ID NO: 5875 cctgaccagacctccgaca7460 7479 SEQ ID NO: 6421 tgtcctacacatggacagg 7617 7636 1 4 SEQ ID NO:5876 agcaagctgcccatcaacg 7667 7686 SEQ ID NO: 6422 cgttgagcaactctttgct7686 7705 1 4 SEQ ID NO: 5877 ggatgaccattaccgggac 7792 7811 SEQ ID NO:6423 gtcccagttggacttatcc 9238 9257 1 4 SEQ ID NO: 5878tggcaaagaatgaggtttt 8028 8047 SEQ ID NO: 6424 aaaaagccctggattgcca 89318950 1 4 SEQ ID NO: 5879 ggcaaagaatgaggttttc 8029 8048 SEQ ID NO: 6425gaaaaagccctggattgcc 8930 8949 1 4 SEQ ID NO: 5880 gggcagcgggtcgagttcc8204 8223 SEQ ID NO: 6426 ggaagaaagcaagctgccc 7660 7679 1 4 SEQ ID NO:5881 gactagctgcggtaatacc 8470 8489 SEQ ID NO: 6427 ggtaccgcccttgcgagtc9091 9110 1 4 SEQ ID NO: 5882 ctcgcgatcccaccacccc 8766 8785 SEQ ID NO:6428 ggggtaccgcccttgcgag 9089 9108 1 4 SEQ ID NO: 5883aggatgattctgatgaccc 8876 8895 SEQ ID NO: 6429 gggtcagcggttgtctcct 24592478 1 4 SEQ ID NO: 5884 agccacttgacctacctca 8976 8995 SEQ ID NO: 6430tgagatcaatagggtggct 9052 9071 1 4 SEQ ID NO: 5885 gggtaccgcccttgcgagt9090 9109 SEQ ID NO: 6431 actcgcgatcccaccaccc 8765 8784 1 4 SEQ ID NO:5886 ctgcaatgactccctccag 1624 1643 SEQ ID NO: 6432 ctggcgggctatggggcag5897 5916 3 3 SEQ ID NO: 5887 ccagcccccgattgggggc 1 20 SEQ ID NO: 6433gcccactggggagtcctgg 1391 1410 2 3 SEQ ID NO: 5888 aaggcgacagcctatcccc520 539 SEQ ID NO: 6434 gggggtctcccccctcctt 6918 6937 2 3 SEQ ID NO:5889 ggccccacggacccccggc 662 681 SEQ ID NO: 6435 gccgcaaagctgtcaggcc4553 4572 2 3 SEQ ID NO: 5890 gaggcggcggacttgatca 983 1002 SEQ ID NO:6436 tgataacatcatgttcctc 8697 8716 2 3 SEQ ID NO: 5891ctgcaattgttcgatctac 1249 1268 SEQ ID NO: 6437 gtaggcggagtcctcgcag 53305349 2 3 SEQ ID NO: 5892 ctccagactgggtttcttg 1637 1656 SEQ ID NO: 6438caagtggcgagctttggag 5599 5618 2 3 SEQ ID NO: 5893 tcgtacctgcgtcgcaggt1830 1849 SEQ ID NO: 6439 acctcagatcattgaacga 8989 9008 2 3 SEQ ID NO:5894 caagacgtgcggggccccc 2026 2045 SEQ ID NO: 6440 gggggagggccgccacttg9156 9175 2 3 SEQ ID NO: 5895 aatgctgcatgcaactgga 2264 2283 SEQ ID NO:6441 tccaggccaataggccatt 9405 9424 2 3 SEQ ID NO: 5896caccctaccggctctgtcc 2383 2402 SEQ ID NO: 6442 ggactacgtccctccggtg 72677286 2 3 SEQ ID NO: 5897 cgccatattacaaggtgtt 2838 2857 SEQ ID NO: 6443aacagccaccaagcaggcg 5554 5573 2 3 SEQ ID NO: 5898 cgaagccatcaagggggga4489 4508 SEQ ID NO: 6444 tcccagatttgggagttcg 8097 8116 2 3 SEQ ID NO:5899 ccagcccgctcaccaccca 5736 5755 SEQ ID NO: 6445 tgggtacaagggagtctgg6382 6401 2 3 SEQ ID NO: 5900 ggctatgactaggtactcc 8635 8654 SEQ ID NO:6446 ggagacatatatcacagcc 9284 9303 2 3 SEQ ID NO: 5901ctccaccatagatcactcc 24 43 SEQ ID NO: 6447 ggagacatcgggccaggag 9111 91301 3 SEQ ID NO: 5902 tccaccatagatcactccc 25 44 SEQ ID NO: 6448gggagttcgatgaaatgga 5451 5470 1 3 SEQ ID NO: 5903 caccatagatcactcccct 2746 SEQ ID NO: 6449 aggggccccaggttgggtg 458 477 1 3 SEQ ID NO: 5904tcactcccctgtgaggaac 36 55 SEQ ID NO: 6450 gttctggaggacggcgtga 809 828 13 SEQ ID NO: 5905 cgttagtatgagtgtcgtg 88 107 SEQ ID NO: 6451cacgctgcacgggccaacg 5191 5210 1 3 SEQ ID NO: 5906 tgtcgtgcagcctccagga100 119 SEQ ID NO: 6452 tcctgttgtcgtggggaca 1879 1898 1 3 SEQ ID NO:5907 ccccccctcccgggagagc 119 138 SEQ ID NO: 6453 gctcccggcctagttgggg 645664 1 3 SEQ ID NO: 5908 ggagagccatagtggtctg 131 150 SEQ ID NO: 6454cagatcattgaacgactcc 8993 9012 1 3 SEQ ID NO: 5909 gagccatagtggtctgcgg134 153 SEQ ID NO: 6455 ccgctgctgggtagcgctc 1048 1067 1 3 SEQ ID NO:5910 gtggtctgcggaaccggtg 142 161 SEQ ID NO: 6456 cacccatatagatgcccac5038 5057 1 3 SEQ ID NO: 5911 agtacaccggaattgccag 161 180 SEQ ID NO:6457 ctggcgggccttgcctact 1406 1425 1 3 SEQ ID NO: 5912ggtcctttcttggatcaac 188 207 SEQ ID NO: 6458 gttgagtgacttcaagacc 63046323 1 3 SEQ ID NO: 5913 ttcttggatcaacccgctc 194 213 SEQ ID NO: 6459gagcggagggggatgagaa 7134 7153 1 3 SEQ ID NO: 5914 ctcaatgcctggagatttg210 229 SEQ ID NO: 6460 caaagactccgacgctgag 7486 7505 1 3 SEQ ID NO:5915 tgcctggagatttgggcgt 215 234 SEQ ID NO: 6461 acgcggccgccgcaaggca1967 1986 1 3 SEQ ID NO: 5916 gcctggagatttgggcgtg 216 235 SEQ ID NO:6462 cacgcggccgccgcaaggc 1966 1985 1 3 SEQ ID NO: 5917gagatttgggcgtgccccc 221 240 SEQ ID NO: 6463 ggggacaaccgatcgtctc 18911910 1 3 SEQ ID NO: 5918 aaaggccttgtggtactgc 273 292 SEQ ID NO: 6464gcagaagaaggtcaccttt 7756 7775 1 3 SEQ ID NO: 5919 aaggccttgtggtactgcc274 293 SEQ ID NO: 6465 ggcagaagaaggtcacctt 7755 7774 1 3 SEQ ID NO:5920 gtggtactgcctgataggg 282 301 SEQ ID NO: 6466 ccctaccggctctgtccac2385 2404 1 3 SEQ ID NO: 5921 cctgatagggtgcttgcga 291 310 SEQ ID NO:6467 tcgccggcccgagggcagg 544 563 1 3 SEQ ID NO: 5922 cgagtgccccgggaggtct307 326 SEQ ID NO: 6468 agacgcagtgtcgcgctcg 4780 4799 1 3 SEQ ID NO:5923 gccccgggaggtctcgtag 312 331 SEQ ID NO: 6469 ctaccttaggttttggggc4122 4141 1 3 SEQ ID NO: 5924 ttacctgttgccgcgcagg 442 461 SEQ ID NO:6470 cctgcgttcgggagggtaa 1023 1042 1 3 SEQ ID NO: 5925tacctgttgccgcgcaggg 443 462 SEQ ID NO: 6471 ccctgcgttcgggagggta 10221041 1 3 SEQ ID NO: 5926 cctgttgccgcgcaggggc 445 464 SEQ ID NO: 6472gcccccgaagccagacagg 8348 8367 1 3 SEQ ID NO: 5927 ctgttgccgcgcaggggcc446 465 SEQ ID NO: 6473 ggcccccgaagccagacag 8347 8366 1 3 SEQ ID NO:5928 tccgagcggtcgcaacccc 497 516 SEQ ID NO: 6474 ggggcaaaggacgtccgga7922 7941 1 3 SEQ ID NO: 5929 ggtcgcaaccccgtggaag 504 523 SEQ ID NO:6475 cttctctgacatggagacc 3268 3287 1 3 SEQ ID NO: 5930gtcgcaaccccgtggaagg 505 524 SEQ ID NO: 6476 ccttcaccattgagacgac 47494768 1 3 SEQ ID NO: 5931 aaggcgacagcctatcccc 520 539 SEQ ID NO: 6477ggggcgctgccagggcctt 774 793 1 3 SEQ ID NO: 5932 cagcctatccccaaggctc 527546 SEQ ID NO: 6478 gagcacaggcttaatgctg 2252 2271 1 3 SEQ ID NO: 5933gagggcagggcctgggctc 554 573 SEQ ID NO: 6479 gagcgtcttcacaggcctc 50205039 1 3 SEQ ID NO: 5934 cagggcctgggctcagccc 559 578 SEQ ID NO: 6480gggcatcggcacagtcctg 4324 4343 1 3 SEQ ID NO: 5935 gggcctgggctcagcccgg561 580 SEQ ID NO: 6481 ccggccgcatatgcggccc 4064 4083 1 3 SEQ ID NO:5936 cctgggctcagcccgggta 564 583 SEQ ID NO: 6482 taccgaccctaacatcagg4162 4181 1 3 SEQ ID NO: 5937 cccctctatggcaatgagg 590 609 SEQ ID NO:6483 cctcgccgacctcatgggg 727 746 1 3 SEQ ID NO: 5938 gagggcatggggtgggcag605 624 SEQ ID NO: 6484 ctgcggatctgttttcctc 1180 1199 1 3 SEQ ID NO:5939 agggcatggggtgggcagg 606 625 SEQ ID NO: 6485 cctgctctttcaccaccct2370 2389 1 3 SEQ ID NO: 5940 aggatggctcctgtcaccc 622 641 SEQ ID NO:6486 gggtcagcggttgtctcct 2459 2478 1 3 SEQ ID NO: 5941gatggctcctgtcaccccg 624 643 SEQ ID NO: 6487 cgggggcgcttacgacatc 42614280 1 3 SEQ ID NO: 5942 tgtcaccccgcggctcccg 633 652 SEQ ID NO: 6488cggggcgcgttccctgaca 3688 3707 1 3 SEQ ID NO: 5943 gtcaccccgcggctcccgg634 653 SEQ ID NO: 6489 ccggggcgcgttccctgac 3687 3706 1 3 SEQ ID NO:5944 gcggctcccggcctagttg 642 661 SEQ ID NO: 6490 caacgtccggggaggccgc2935 2954 1 3 SEQ ID NO: 5945 ctcccggcctagttggggc 646 665 SEQ ID NO:6491 gccctgtcgaacactggag 4439 4458 1 3 SEQ ID NO: 5946ataccctcacatgcggcct 711 730 SEQ ID NO: 6492 aggcaacattatcatgtat 88398858 1 3 SEQ ID NO: 5947 ttccgctcgtcggcggccc 750 769 SEQ ID NO: 6493gggcaaagcacatgtggaa 5625 5644 1 3 SEQ ID NO: 5948 cccctagggggcgctgcca767 786 SEQ ID NO: 6494 tggcaatgagggcatgggg 598 617 1 3 SEQ ID NO: 5949tgcaacagggaacctgccc 832 851 SEQ ID NO: 6495 gggctcattcgtgcatgca 30923111 1 3 SEQ ID NO: 5950 gcgtaacgcgtccggggta 922 941 SEQ ID NO: 6496taccaccacgagcttacgc 2751 2770 1 3 SEQ ID NO: 5951 tcaagcattgtgtttgagg968 987 SEQ ID NO: 6497 cctctatgcccccccttga 7512 7531 1 3 SEQ ID NO:5952 cccacgctcgcggccagga 1070 1089 SEQ ID NO: 6498 tcctgtttaacatcttggg5763 5782 1 3 SEQ ID NO: 5953 cggccaggaatgctaccat 1080 1099 SEQ ID NO:6499 atggcatgcatgtcggccg 5279 5298 1 3 SEQ ID NO: 5954acgacaatacgacaccacg 1106 1125 SEQ ID NO: 6500 cgtggggacaaccgatcgt 18881907 1 3 SEQ ID NO: 5955 gggcggctgctctctgctc 1140 1159 SEQ ID NO: 6501gagcaacttgaaaaagccc 8921 8940 1 3 SEQ ID NO: 5956 cgtgggggacctctgcgga1168 1187 SEQ ID NO: 6502 tccgttgccggagcgcacg 2615 2634 1 3 SEQ ID NO:5957 agctgttcaccttctcgcc 1206 1225 SEQ ID NO: 6503 ggcgacaatagagggagct3779 3798 1 3 SEQ ID NO: 5958 ctgttcaccttctcgcccc 1208 1227 SEQ ID NO:6504 ggggagacatatatcacag 9282 9301 1 3 SEQ ID NO: 5959ctgcaattgttcgatctac 1249 1268 SEQ ID NO: 6505 gtaggactggcaggggcag 48094828 1 3 SEQ ID NO: 5960 attgttcgatctaccccgg 1254 1273 SEQ ID NO: 6506ccggcccaaaaggcccaat 3615 3634 1 3 SEQ ID NO: 5961 atctaccccggccacgcgt1262 1281 SEQ ID NO: 6507 acgccatggaccgggagat 2766 2785 1 3 SEQ ID NO:5962 cggccacgcgtcaggtcac 1270 1289 SEQ ID NO: 6508 gtgatgctactttttgccg1460 1479 1 3 SEQ ID NO: 5963 ccgcatggcctgggacatg 1288 1307 SEQ ID NO:6509 catggaaactactatgcgg 3943 3962 1 3 SEQ ID NO: 5964cgcagttactccggatccc 1344 1363 SEQ ID NO: 6510 gggaacccaggaggatgcg 85938612 1 3 SEQ ID NO: 5965 cccacaagccgtcatcgac 1360 1379 SEQ ID NO: 6511gtcgtcaccagcacctggg 5306 5325 1 3 SEQ ID NO: 5966 ctggggagtcctggcgggc1396 1415 SEQ ID NO: 6512 gcccggagcgcatggccag 1695 1714 1 3 SEQ ID NO:5967 ggcgggccttgcctactat 1408 1427 SEQ ID NO: 6513 atagaagaagcctgccgcc7865 7884 1 3 SEQ ID NO: 5968 tttgccggcgttgacgggc 1472 1491 SEQ ID NO:6514 gcccccacattcggccaaa 7888 7907 1 3 SEQ ID NO: 5969caccctcacaacggggggg 1492 1511 SEQ ID NO: 6515 ccccaatatcgaggaggtg 44204439 1 3 SEQ ID NO: 5970 gggggggcacgctgcccgc 1504 1523 SEQ ID NO: 6516gcggcacggcgaccgcccc 7440 7459 1 3 SEQ ID NO: 5971 ggggcacgctgcccgcctc1507 1526 SEQ ID NO: 6517 gagggagcttgctctcccc 3789 3808 1 3 SEQ ID NO:5972 gcccgcctcaccagcgggt 1517 1536 SEQ ID NO: 6518 accctcacaacgggggggc1493 1512 1 3 SEQ ID NO: 5973 atccagcttataaacacca 1571 1590 SEQ ID NO:6519 tggttatcgtgggtaggat 5382 5401 1 3 SEQ ID NO: 5974ctccagactgggtttcttg 1637 1656 SEQ ID NO: 6520 caagcggagacggctggag 43464365 1 3 SEQ ID NO: 5975 cccggagcgcatggccagc 1696 1715 SEQ ID NO: 6521gctgtgggcgtcttccggg 3869 3888 1 3 SEQ ID NO: 5976 ctgccgctccattgacaag1714 1733 SEQ ID NO: 6522 cttggtacatcaagggcag 2667 2686 1 3 SEQ ID NO:5977 aagttcgaccagggatggg 1730 1749 SEQ ID NO: 6523 cccaaccagaatacgactt8673 8692 1 3 SEQ ID NO: 5978 ggggtcctatcacttatgc 1746 1765 SEQ ID NO:6524 gcatgtgtgggttcccccc 2914 2933 1 3 SEQ ID NO: 5979ccagaggccttattgctgg 1786 1805 SEQ ID NO: 6525 ccaggatctcgtcggctgg 36583677 1 3 SEQ ID NO: 5980 cccacctcaacaatgtggt 1810 1829 SEQ ID NO: 6526accaagatcatcacctggg 3284 3303 1 3 SEQ ID NO: 5981 tcgtacctgcgtcgcaggt1830 1849 SEQ ID NO: 6527 accttcaccattgagacga 4748 4767 1 3 SEQ ID NO:5982 tgcgtcgcaggtgtgtggt 1837 1856 SEQ ID NO: 6528 accatgtctcccccacgca6123 6142 1 3 SEQ ID NO: 5983 tggggacaaccgatcgtct 1890 1909 SEQ ID NO:6529 agacgacgaccgtgcccca 4761 4780 1 3 SEQ ID NO: 5984cagctggggggagaacgat 1924 1943 SEQ ID NO: 6530 atcggagctcagcccgctg 23202339 1 3 SEQ ID NO: 5985 cgccgcaaggcaactggtt 1974 1993 SEQ ID NO: 6531aacccaggaggatgcggcg 8596 8615 1 3 SEQ ID NO: 5986 gccgcaaggcaactggttc1975 1994 SEQ ID NO: 6532 gaacccaggaggatgcggc 8595 8614 1 3 SEQ ID NO:5987 ctgtacatggatgaatagc 1996 2015 SEQ ID NO: 6533 gctataaaatcgctcacag8366 8385 1 3 SEQ ID NO: 5988 tgtacatggatgaatagca 1997 2016 SEQ ID NO:6534 tgctgctcaatgtcctaca 7607 7626 1 3 SEQ ID NO: 5989gttcaccaagacgtgcggg 2020 2039 SEQ ID NO: 6535 cccgctcaccacccagaac 57405759 1 3 SEQ ID NO: 5990 agacgtgcggggccccccc 2028 2047 SEQ ID NO: 6536ggggaggttcaagtggtct 3512 3531 1 3 SEQ ID NO: 5991 cccccgtgtaacatcgggg2042 2061 SEQ ID NO: 6537 ccccaatcgatgaacgggg 9376 9395 1 3 SEQ ID NO:5992 taacaccttgacctgcccc 2071 2090 SEQ ID NO: 6538 ggggacgaccttgtcgtta8561 8580 1 3 SEQ ID NO: 5993 ggctctggcactacccctg 2184 2203 SEQ ID NO:6539 caggaggatgcggcgagcc 8600 8619 1 3 SEQ ID NO: 5994tgcactgtcaacttctcca 2201 2220 SEQ ID NO: 6540 tggatggggtgcggttgca 67176736 1 3 SEQ ID NO: 5995 caggcttaatgctgcatgc 2257 2276 SEQ ID NO: 6541gcatcatgcacaccacctg 6411 6430 1 3 SEQ ID NO: 5996 aatgctgcatgcaactgga2264 2283 SEQ ID NO: 6542 tccatggtcttagcgcatt 9009 9028 1 3 SEQ ID NO:5997 ctgcatgcaactggacccg 2268 2287 SEQ ID NO: 6543 cgggaccttgcggtagcag3236 3255 1 3 SEQ ID NO: 5998 caactggacccgaggagag 2275 2294 SEQ ID NO:6544 ctcttacgggatgaggttg 6761 6780 1 3 SEQ ID NO: 5999gacagggacagatcggagc 2309 2328 SEQ ID NO: 6545 gctctcccccaggcctgtc 37993818 1 3 SEQ ID NO: 6000 gacagatcggagctcagcc 2315 2334 SEQ ID NO: 6546ggctggagcgcggcttgtc 4357 4376 1 3 SEQ ID NO: 6001 acagatcggagctcagccc2316 2335 SEQ ID NO: 6547 gggccaacgcccctgctgt 5201 5220 1 3 SEQ ID NO:6002 actggcttgatccacctcc 2402 2421 SEQ ID NO: 6548 ggagagggggccgtgcagt6068 6087 1 3 SEQ ID NO: 6003 ggcttgatccacctccatc 2405 2424 SEQ ID NO:6549 gatgatgctgctgatagcc 2551 2570 1 3 SEQ ID NO: 6004gtcagcggttgtctccttt 2461 2480 SEQ ID NO: 6550 aaaggacggttgtcctgac 73447363 1 3 SEQ ID NO: 6005 gagtatgtcgtgttgcttt 2492 2511 SEQ ID NO: 6551aaagaccaagctcaaactc 9202 9221 1 3 SEQ ID NO: 6006 tgtggatgatgctgctgat2547 2566 SEQ ID NO: 6552 atcactgatggcattcaca 5707 5726 1 3 SEQ ID NO:6007 ccgaggccgccttagagaa 2574 2593 SEQ ID NO: 6553 ttctgattgccatactcgg3015 3034 1 3 SEQ ID NO: 6008 agaacctggtggccctcaa 2589 2608 SEQ ID NO:6554 ttgatatcaccaaacttct 3000 3019 1 3 SEQ ID NO: 6009tacatcaagggcaggctgg 2672 2691 SEQ ID NO: 6555 ccagatgtacactaatgta 36373656 1 3 SEQ ID NO: 6010 caagggcaggctggtccct 2677 2696 SEQ ID NO: 6556aggggtaggcatctacttg 9355 9374 1 3 SEQ ID NO: 6011 gcatggccgctgctcctgc2720 2739 SEQ ID NO: 6557 gcagtgctcacttccatgc 6848 6867 1 3 SEQ ID NO:6012 catggccgctgctcctgct 2721 2740 SEQ ID NO: 6558 agcagtgctcacttccatg6847 6866 1 3 SEQ ID NO: 6013 gccgctgctcctgctcctc 2725 2744 SEQ ID NO:6559 gagggccgccacttgcggc 9160 9179 1 3 SEQ ID NO: 6014ggagatggctgcatcgtgc 2779 2798 SEQ ID NO: 6560 gcacggcgaccgcccctcc 74437462 1 3 SEQ ID NO: 6015 atggctgcatcgtgcggag 2783 2802 SEQ ID NO: 6561ctccaggccaataggccat 9404 9423 1 3 SEQ ID NO: 6016 ggcgcggtttttgtgggtc2801 2820 SEQ ID NO: 6562 gaccattaccacgggcgcc 4192 4211 1 3 SEQ ID NO:6017 tcttatcaccagagctgag 2887 2906 SEQ ID NO: 6563 ctcacaggccgggacaaga3482 3501 1 3 SEQ ID NO: 6018 gtgtgggttccccccctca 2918 2937 SEQ ID NO:6564 tgaggtcaccctcacacac 5242 5261 1 3 SEQ ID NO: 6019tccccccctcaacgtccgg 2926 2945 SEQ ID NO: 6565 ccggctcgtggctgaggga 62616280 1 3 SEQ ID NO: 6020 ctcaacgtccggggaggcc 2933 2952 SEQ ID NO: 6566ggcctgttactccattgag 8959 8978 1 3 SEQ ID NO: 6021 accaaacttctgattgcca3008 3027 SEQ ID NO: 6567 tggctctctacgatgtggt 8130 8149 1 3 SEQ ID NO:6022 caaacttctgattgccata 3010 3029 SEQ ID NO: 6568 tatgacacccgctgttttg8267 8286 1 3 SEQ ID NO: 6023 ggaccgctcatggtgctcc 3032 3051 SEQ ID NO:6569 ggagatcctgcggaagtcc 7171 7190 1 3 SEQ ID NO: 6024gaccgctcatggtgctcca 3033 3052 SEQ ID NO: 6570 tggaaactactatgcggtc 39453964 1 3 SEQ ID NO: 6025 atgcatgttagtgcggaaa 3106 3125 SEQ ID NO: 6571tttctgtaggggtaggcat 9348 9367 1 3 SEQ ID NO: 6026 ttatgtccaaatggccttc3139 3158 SEQ ID NO: 6572 gaagccagacaggctataa 8354 8373 1 3 SEQ ID NO:6027 ccaaatggccttcatgaga 3145 3164 SEQ ID NO: 6573 tctcagcgacgggtcttgg7552 7571 1 3 SEQ ID NO: 6028 ccttcatgagactgggcgc 3153 3172 SEQ ID NO:6574 gcgctcgtggccttcaagg 5927 5946 1 3 SEQ ID NO: 6029ccttgcggtagcagtggag 3241 3260 SEQ ID NO: 6575 ctccgcccgaaggggaagg 33493368 1 3 SEQ ID NO: 6030 tgtcgtcttctctgacatg 3262 3281 SEQ ID NO: 6576catggtctacgccacgaca 7717 7736 1 3 SEQ ID NO: 6031 tggggggcagacaccgcgg3299 3318 SEQ ID NO: 6577 ccgccttatcgtattccca 8083 8102 1 3 SEQ ID NO:6032 ggggggcagacaccgcggc 3300 3319 SEQ ID NO: 6578 gccgcccaactcgctcccc5792 5811 1 3 SEQ ID NO: 6033 gtggggacatcatcctggg 3321 3340 SEQ ID NO:6579 cccatctacacgctcccac 4020 4039 1 3 SEQ ID NO: 6034tggggacatcatcctgggc 3322 3341 SEQ ID NO: 6580 gcccatctacacgctccca 40194038 1 3 SEQ ID NO: 6035 ggggacatcatcctgggcc 3323 3342 SEQ ID NO: 6581ggccagggggtctcccccc 6913 6932 1 3 SEQ ID NO: 6036 acctgtctccgcccgaagg3343 3362 SEQ ID NO: 6582 cctttgacagactgcaggt 7770 7789 1 3 SEQ ID NO:6037 tgtctccgcccgaagggga 3346 3365 SEQ ID NO: 6583 tccccggtcttcacagaca3962 3981 1 3 SEQ ID NO: 6038 gggagatactcctggggcc 3366 3385 SEQ ID NO:6584 ggcccatctacacgctccc 4018 4037 1 3 SEQ ID NO: 6039ctcccaacagacccggggc 3439 3458 SEQ ID NO: 6585 gcccccccttgagggggag 75197538 1 3 SEQ ID NO: 6040 tccaccgcaacacaatctt 3530 3549 SEQ ID NO: 6586aagaggctccaccagtgga 6215 6234 1 3 SEQ ID NO: 6041 cacaatctttcctggcgac3540 3559 SEQ ID NO: 6587 gtcgtcggagtcgtgtgtg 6020 6039 1 3 SEQ ID NO:6042 ggctggccggcgccccccg 3671 3690 SEQ ID NO: 6588 cgggttgttgcaaacagcc5542 5561 1 3 SEQ ID NO: 6043 ccccggggcgcgttccctg 3685 3704 SEQ ID NO:6589 caggtttgtaactccgggg 4840 4859 1 3 SEQ ID NO: 6044tccctgacaccatgcacct 3698 3717 SEQ ID NO: 6590 aggtcacgcgggtggggga 66186637 1 3 SEQ ID NO: 6045 ttccggtgcgccggcgggg 3762 3781 SEQ ID NO: 6591ccccgttgagtccatggaa 3931 3950 1 3 SEQ ID NO: 6046 ctcccccaggcctgtctcc3802 3821 SEQ ID NO: 6592 ggagacatcgggccaggag 9111 9130 1 3 SEQ ID NO:6047 gggggttgcaaaggcggtg 3904 3923 SEQ ID NO: 6593 caccctgcctgggaacccc5680 5699 1 3 SEQ ID NO: 6048 tttgtccccgttgagtcca 3926 3945 SEQ ID NO:6594 tggagaccttctgggcaaa 5613 5632 1 3 SEQ ID NO: 6049ccgtaccgcaaacattcca 3996 4015 SEQ ID NO: 6595 tggattgccaaatctacgg 89408959 1 3 SEQ ID NO: 6050 caagtggcccatctacacg 4013 4032 SEQ ID NO: 6596cgtgggtaggatcatcttg 5389 5408 1 3 SEQ ID NO: 6051 cacgctcccactggcagcg4028 4047 SEQ ID NO: 6597 cgctgcttcggctttcgtg 5815 5834 1 3 SEQ ID NO:6052 ccgcatatgcggcccaagg 4068 4087 SEQ ID NO: 6598 ccttcaaggtcatgagcgg5937 5956 1 3 SEQ ID NO: 6053 cgtatatgtctaaagcaca 4140 4159 SEQ ID NO:6599 tgtggaagtgtctcatacg 5163 5182 1 3 SEQ ID NO: 6054gtatatgtctaaagcacat 4141 4160 SEQ ID NO: 6600 atgtggaagtgtctcatac 51625181 1 3 SEQ ID NO: 6055 ggaccattaccacgggcgc 4191 4210 SEQ ID NO: 6601gcgcgtgtcactcaggtcc 6167 6186 1 3 SEQ ID NO: 6056 cccccattacgtactccac4209 4228 SEQ ID NO: 6602 gtgggcccgggagaggggg 6059 6078 1 3 SEQ ID NO:6057 agttccttgccgacggtgg 4236 4255 SEQ ID NO: 6603 ccacagtcaaggctaaact7839 7858 1 3 SEQ ID NO: 6058 gagacggctggagcgcggc 4352 4371 SEQ ID NO:6604 gccgggggaccccgatctc 7537 7556 1 3 SEQ ID NO: 6059caccgctacgcctccagga 4384 4403 SEQ ID NO: 6605 tcctacacatggacaggtg 76197638 1 3 SEQ ID NO: 6060 tggagagatccccttctac 4453 4472 SEQ ID NO: 6606gtagcagtgctcacttcca 6845 6864 1 3 SEQ ID NO: 6061 agccatccccatcgaagcc4477 4496 SEQ ID NO: 6607 ggctggttcgttgctggct 9257 9276 1 3 SEQ ID NO:6062 tccccatcgaagccatcaa 4482 4501 SEQ ID NO: 6608 ttgagggggagccggggga7527 7546 1 3 SEQ ID NO: 6063 ccccatcgaagccatcaag 4483 4502 SEQ ID NO:6609 cttgagggggagccggggg 7526 7545 1 3 SEQ ID NO: 6064ggcctcggaatcaatgctg 4568 4587 SEQ ID NO: 6610 cagctccgaattgtcggcc 74147433 1 3 SEQ ID NO: 6065 gtccgtcataccgaccagc 4612 4631 SEQ ID NO: 6611gctgagggatgtttgggac 6271 6290 1 3 SEQ ID NO: 6066 gtcataccgaccagcggag4616 4635 SEQ ID NO: 6612 ctccattgagccacttgac 8968 8987 1 3 SEQ ID NO:6067 cgggctataccggtgactt 4668 4687 SEQ ID NO: 6613 aagtccaagaagttccccg7184 7203 1 3 SEQ ID NO: 6068 ctttgattcagtgatcgac 4684 4703 SEQ ID NO:6614 gtcgagttcctggtaaaag 8213 8232 1 3 SEQ ID NO: 6069acagtcgacttcagcttgg 4724 4743 SEQ ID NO: 6615 ccaaatctacggggcctgt 89478966 1 3 SEQ ID NO: 6070 cttggaccccaccttcacc 4738 4757 SEQ ID NO: 6616ggtgttgagtgacttcaag 6301 6320 1 3 SEQ ID NO: 6071 gagacgacgaccgtgcccc4760 4779 SEQ ID NO: 6617 ggggacaaccgatcgtctc 1891 1910 1 3 SEQ ID NO:6072 ggggtaggactggcagggg 4806 4825 SEQ ID NO: 6618 ccccccggggacttgcccc8657 8676 1 3 SEQ ID NO: 6073 gggcatatacaggtttgta 4831 4850 SEQ ID NO:6619 tacacatggacaggtgccc 7622 7641 1 3 SEQ ID NO: 6074gggggaacggccctcgggc 4855 4874 SEQ ID NO: 6620 gcccctgcacgccttcccc 65466565 1 3 SEQ ID NO: 6075 tgacgcgggctgtgcttgg 4906 4925 SEQ ID NO: 6621ccaattgacaccaccgtca 8009 8028 1 3 SEQ ID NO: 6076 gacgcgggctgtgcttggt4907 4926 SEQ ID NO: 6622 accaattgacaccaccgtc 8008 8027 1 3 SEQ ID NO:6077 tgcttggtacgagctcacc 4918 4937 SEQ ID NO: 6623 ggtgcggctgttggcagca5849 5868 1 3 SEQ ID NO: 6078 tgcccacttcctgtcccag 5050 5069 SEQ ID NO:6624 ctgggcgcgctgacgggca 3164 3183 1 3 SEQ ID NO: 6079ggtggcataccaagccaca 5101 5120 SEQ ID NO: 6625 tgtgacaccaattgacacc 80028021 1 3 SEQ ID NO: 6080 gggctcaggccccacctcc 5130 5149 SEQ ID NO: 6626ggaggccgcaagccagccc 8066 8085 1 3 SEQ ID NO: 6081 ccatcgtgggatcaaatgt5147 5166 SEQ ID NO: 6627 acattctggcgggctatgg 5892 5911 1 3 SEQ ID NO:6082 tcatacggctaaaacccac 5175 5194 SEQ ID NO: 6628 gtggccttcaaggtcatga5933 5952 1 3 SEQ ID NO: 6083 tgctgtataggctaggggc 5214 5233 SEQ ID NO:6629 gcccgaaccggacgtagca 6832 6851 1 3 SEQ ID NO: 6084ccaaatacatcatggcatg 5268 5287 SEQ ID NO: 6630 catgcctcaggaaacttgg 90729091 1 3 SEQ ID NO: 6085 ggagtcctcgcagctctgg 5336 5355 SEQ ID NO: 6631ccagctgtctgcgccctcc 6955 6974 1 3 SEQ ID NO: 6086 gcctgacaacaggcagtgt5364 5383 SEQ ID NO: 6632 acactccaggccaataggc 9401 9420 1 3 SEQ ID NO:6087 agccaccaagcaggcggag 5557 5576 SEQ ID NO: 6633 ctccagttaactcctggct8820 8839 1 3 SEQ ID NO: 6088 catgtggaatttcatcagc 5635 5654 SEQ ID NO:6634 gctgcgccatcacaacatg 7702 7721 1 3 SEQ ID NO: 6089ctctatcaccagcccgctc 5728 5747 SEQ ID NO: 6635 gagccgcatgactgcagag 95659584 1 3 SEQ ID NO: 6090 cccagaacaccctcctgtt 5751 5770 SEQ ID NO: 6636aacatcttgggggggtggg 5771 5790 1 3 SEQ ID NO: 6091 ctcctgtttaacatcttgg5762 5781 SEQ ID NO: 6637 ccaatcgatgaacggggag 9378 9397 1 3 SEQ ID NO:6092 ttgggggggtgggtagccg 5777 5796 SEQ ID NO: 6638 cggcgccaaactattccaa6564 6583 1 3 SEQ ID NO: 6093 tgcttcggctttcgtgggc 5818 5837 SEQ ID NO:6639 gcccgaaccggacgtagca 6832 6851 1 3 SEQ ID NO: 6094tcgtgggcgctggtatcgc 5829 5848 SEQ ID NO: 6640 gcgagcggcgtgctgacga 84538472 1 3 SEQ ID NO: 6095 cgctggtgcggctgttggc 5845 5864 SEQ ID NO: 6641gccacgacatcccgcagcg 7727 7746 1 3 SEQ ID NO: 6096 cggctgttggcagcatagg5853 5872 SEQ ID NO: 6642 cctagactctttcgagccg 7111 7130 1 3 SEQ ID NO:6097 ggggcaggggtggctggcg 5909 5928 SEQ ID NO: 6643 cgcccaactcgctcccccc5794 5813 1 3 SEQ ID NO: 6098 ctggcgcgctcgtggcctt 5922 5941 SEQ ID NO:6644 aagggaggccgcaagccag 8063 8082 1 3 SEQ ID NO: 6099tggcgcgctcgtggccttc 5923 5942 SEQ ID NO: 6645 gaagggaggccgcaagcca 80628081 1 3 SEQ ID NO: 6100 gagcggcgaggcgccctct 5950 5969 SEQ ID NO: 6646agagcgtcgtctgctgctc 7596 7615 1 3 SEQ ID NO: 6101 tgggcccgggagagggggc6060 6079 SEQ ID NO: 6647 gcccatctacacgctccca 4019 4038 1 3 SEQ ID NO:6102 cggctgatagcgttcgctt 6095 6114 SEQ ID NO: 6648 aagcaggcggaggctgccg5564 5583 1 3 SEQ ID NO: 6103 gtgcctgagagcgacgccg 6146 6165 SEQ ID NO:6649 cggccgccgacagcggcac 7428 7447 1 3 SEQ ID NO: 6104atgaggactgttctacgcc 6237 6256 SEQ ID NO: 6650 ggcggggggacggcatcat 63996418 1 3 SEQ ID NO: 6105 gtccaagctcctgccgcgg 6331 6350 SEQ ID NO: 6651ccgctccgtgtgggaggac 7969 7988 1 3 SEQ ID NO: 6106 acagatcgccggacatgtc6442 6461 SEQ ID NO: 6652 gacatatatcacagcctgt 9287 9306 1 3 SEQ ID NO:6107 acgtggcatggaacattcc 6506 6525 SEQ ID NO: 6653 ggaagaacccggactacgt7257 7276 1 3 SEQ ID NO: 6108 gggcccctgcacgccttcc 6544 6563 SEQ ID NO:6654 ggaagaaagcaagctgccc 7660 7679 1 3 SEQ ID NO: 6109agtgcccatgtcaggttcc 6675 6694 SEQ ID NO: 6655 ggaaacagctagacacact 88038822 1 3 SEQ ID NO: 6110 tgcccatgtcaggttccag 6677 6696 SEQ ID NO: 6656ctgggcgcgctgacgggca 3164 3183 1 3 SEQ ID NO: 6111 cagctcctgagtttttcac6693 6712 SEQ ID NO: 6657 gtgagagcgtcgtctgctg 7593 7612 1 3 SEQ ID NO:6112 tcacggaggtggatggggt 6708 6727 SEQ ID NO: 6658 acccttcctcaagccgtga8153 8172 1 3 SEQ ID NO: 6113 cacggaggtggatggggtg 6709 6728 SEQ ID NO:6659 cacccttcctcaagccgtg 8152 8171 1 3 SEQ ID NO: 6114gacccctcccacattacag 6872 6891 SEQ ID NO: 6660 ctgttttgactcaacggtc 82788297 1 3 SEQ ID NO: 6115 ttggccagggggtctcccc 6911 6930 SEQ ID NO: 6661ggggtgggtagccgcccaa 5782 5801 1 3 SEQ ID NO: 6116 ccttgagggcgacatgcac6972 6991 SEQ ID NO: 6662 gtgcttaaggagatgaagg 7811 7830 1 3 SEQ ID NO:6117 ggagatgggcggaaacatc 7060 7079 SEQ ID NO: 6663 gatgacccatttcttctcc8887 8906 1 3 SEQ ID NO: 6118 gagatgggcggaaacatca 7061 7080 SEQ ID NO:6664 tgatgacccatttcttctc 8886 8905 1 3 SEQ ID NO: 6119ctagactctttcgagccgc 7112 7131 SEQ ID NO: 6665 gcggcgtgctgacgactag 84578476 1 3 SEQ ID NO: 6120 tagactctttcgagccgct 7113 7132 SEQ ID NO: 6666agcgacgggtcttggtcta 7556 7575 1 3 SEQ ID NO: 6121 agaatgaaatatccattgc7149 7168 SEQ ID NO: 6667 gcaaagaatgaggttttct 8030 8049 1 3 SEQ ID NO:6122 ttgcggcggagatcctgcg 7164 7183 SEQ ID NO: 6668 cgcacgatgcatctggcaa8730 8749 1 3 SEQ ID NO: 6123 agcgaggaggctggtgaga 7580 7599 SEQ ID NO:6669 tctcgtgcccgaccccgct 9305 9324 1 3 SEQ ID NO: 6124tgagagcgtcgtctgctgc 7594 7613 SEQ ID NO: 6670 gcagtaaagaccaagctca 91979216 1 3 SEQ ID NO: 6125 gtcgtctgctgctcaatgt 7601 7620 SEQ ID NO: 6671acatggtctacgccacgac 7716 7735 1 3 SEQ ID NO: 6126 tgcgccatcacaacatggt7704 7723 SEQ ID NO: 6672 accatgtctcccccacgca 6123 6142 1 3 SEQ ID NO:6127 cagaagaaggtcacctttg 7757 7776 SEQ ID NO: 6673 caaagaatgaggttttctg8031 8050 1 3 SEQ ID NO: 6128 cctggatgaccattaccgg 7789 7808 SEQ ID NO:6674 ccggaacctatccagcagg 7936 7955 1 3 SEQ ID NO: 6129ggacgtgcttaaggagatg 7807 7826 SEQ ID NO: 6675 catcgggccaggagcgtcc 91169135 1 3 SEQ ID NO: 6130 aaagaatgaggttttctgc 8032 8051 SEQ ID NO: 6676gcagaagaaggtcaccttt 7756 7775 1 3 SEQ ID NO: 6131 agttcgtgtatgcgagaag8110 8129 SEQ ID NO: 6677 cttcatgcctcaggaaact 9069 9088 1 3 SEQ ID NO:6132 ggctataaaatcgctcaca 8365 8384 SEQ ID NO: 6678 tgtgaaaggtccgtgagcc9551 9570 1 3 SEQ ID NO: 6133 ttctccatccttctagctc 8900 8919 SEQ ID NO:6679 gagcggagggggatgagaa 7134 7153 1 3 SEQ ID NO: 6134tgtctcgtgcccgaccccg 9303 9322 SEQ ID NO: 6680 cggggcgcgttccctgaca 36883707 1 3

TABLE 14 Sequences from human hepatitis C virus (HCV) (Direct MatchType) Start End Start End Source Index Index Match Index Index Match #SEQ ID NO: ttttttttttttttttttt 9446 9465 SEQ ID NO: 4661ttttttttttttttttttt 9466 9485 2 4088 SEQ ID NO: ttttttttttttttttttt 94469465 SEQ ID NO: 5229 ttttttttttttttttttt 9465 9484 1 4347 SEQ ID NO:ttttttttttttttttttt 9447 9466 SEQ ID NO: 5230 ttttttttttttttttttt 94669485 1 4348

TABLE 15 Sequences of Exemplary Gene Targetsgi|4502152|ref|NM_000384.1| Homo sapiens apolipoprotein B (includingAg(x) antigen) (APOB), mRNAATTCCCACCGGGACCTGCGGGGCTGAGTGCCCTTCTCGGTTGCTGCCGCTGAGGAGCCCGCCCAGCCAGCCAGGGCCGCGAGGCCGAGGCCAGGCCGCAGCCCAGGAGCCGCCCCACCGCAGCTGGCGATGGACCCGCCGAGGCCCGCGCTGCTGGCGCTGCTGGCGCTGCCTGCGCTGCTGCTGCTGCTGCTGGCGGGCGCCAGGGCCGAAGAGGAAATGCTGGAAAATGTCAGCCTGGTCTGTCCAAAAGATGCGACCCGATTCAAGCACCTCCGGAAGTACACATACAACTATGAGGCTGAGAGTTCCAGTGGAGTCCCTGGGACTGCTGATTCAAGAAGTGCCACCAGGATCAACTGCAAGGTTGAGCTGGAGGTTCCCCAGCTCTGCAGCTTCATCCTGAAGACCAGCCAGTGCACCCTGAAAGAGGTGTATGGCTTCAACCCTGAGGGCAAAGCCTTGCTGAAGAAAACCAAGAACTCTGAGGAGTTTGCTGCAGCCATGTCCAGGTATGAGCTCAAGCTGGCCATTCCAGAAGGGAAGCAGGTTTTCCTTTACCCGGAGAAAGATGAACCTACTTACATCCTGAACATCAAGAGGGGCATCATTTCTGCCCTCCTGGTTCCCCCAGAGACAGAAGAAGCCAAGCAAGTGTTGTTTCTGGATACCGTGTATGGAAACTGCTCCACTCACTTTACCGTCAAGACGAGGAAGGGCAATGTGGCAACAGAAATATCCACTGAAAGAGACCTGGGGCAGTGTGATCGCTTCAAGCCCATCCGCACAGGCATCAGCCCACTTGCTCTCATCAAAGGCATGACCCGCCCCTTGTCAACTCTGATCAGCAGCAGCCAGTCCTGTCAGTACACACTGGACGCTAAGAGGAAGCATGTGGCAGAAGCCATCTGCAAGGAGCAACACCTCTTCCTGCCTTTCTCCTACAACAATAAGTATGGGATGGTAGCACAAGTGACACAGACTTTGAAACTTGAAGACACACCAAAGATCAACAGCCGCTTCTTTGGTGAAGGTACTAAGAAGATGGGCCTCGCATTTGAGAGCACCAAATCCACATCACCTCCAAAGCAGGCCGAAGCTGTTTTGAAGACTCTCCAGGAACTGAAAAAACTAACCATCTCTGAGCAAAATATCCAGAGAGCTAATCTCTTCAATAAGCTGGTTACTGAGCTGAGAGGCCTCAGTGATGAAGCAGTCACATCTCTCTTGCCACAGCTGATTGAGGTGTCCAGCCCCATCACTTTACAAGCCTTGGTTCAGTGTGGACAGCCTCAGTGCTCCACTCACATCCTCCAGTGGCTGAAACGTGTGCATGCCAACCCCCTTCTGATAGATGTGGTCACCTACCTGGTGGCCCTGATCCCCGAGCCCTCAGCACAGCAGCTGCGAGAGATCTTCAACATGGCGAGGGATCAGCGCAGCCGAGCCACCTTGTATGCGCTGAGCCACGCGGTCAACAACTATCATAAGACAAACCCTACAGGGACCCAGGAGCTGCTGGACATTGCTAATTACCTGATGGAACAGATTCAAGATGACTGCACTGGGGATGAAGATTACACCTATTTGATTCTGCGGGTCATTGGAAATATGGGCCAAACCATGGAGCAGTTAACTCCAGAACTCAAGTCTTCAATCCTCAAATGTGTCCAAAGTACAAAGCCATCACTGATGATCCAGAAAGCTGCCATCCAGGCTCTGCGGAAAATGGAGCCTAAAGACAAGGACCAGGAGGTTCTTCTTCAGACTTTCCTTGATGATGCTTCTCCGGGAGATAAGCGACTGGCTGCCTATCTTATGTTGATGAGGAGTCCTTCACAGGCAGATATTAACAAAATTGTCCAAATTCTACCATGGGAACAGAATGAGCAAGTGAAGAACTTTGTGGCTTCCCATATTGCCAATATCTTGAACTCAGAAGAATTGGATATCCAAGATCTGAAAAAGTTAGTGAAAGAAGCTCTGAAAGAATCTCAACTTCCAACTGTCATGGACTTCAGAAAATTCTCTCGGAACTATCAACTCTACAAATCTGTTTCTCTTCCATCACTTGACCCAGCCTCAGCCAAAATAGAAGGGAATCTTATATTTGATCCAAATAACTACCTTCCTAAAGAAAGCATGCTGAAAACTACCCTCACTGCCTTTGGATTTGCTTCAGCTGACCTCATCGAGATTGGCTTGGAAGGAAAAGGCTTTGAGCCAACATTGGAAGCTCTTTTTGGGAAGCAAGGATTTTTCCCAGACAGTGTCAACAAAGCTTTGTACTGGGTTAATGGTCAAGTTCCTGATGGTGTCTCTAAGGTCTTAGTGGACCACTTTGGCTATACCAAAGATGATAAACATGAGCAGGATATGGTAAATGGAATAATGCTCAGTGTTGAGAAGCTGATTAAAGATTTGAAATCCAAAGAAGTCCCGGAAGCCAGAGCCTACCTCCGCATCTTGGGAGAGGAGCTTGGTTTTGCCAGTCTCCATGACCTCCAGCTCCTGGGAAAGCTGCTTCTGATGGGTGCCCGCACTCTGCAGGGGATCCCCCAGATGATTGGAGAGGTCATCAGGAAGGGCTCAAAGAATGACTTTTTTCTTCACTACATCTTCATGGAGAATGCCTTTGAACTCCCCACTGGAGCTGGATTACAGTTGCAAATATCTTCATCTGGAGTCATTGCTCCCGGAGCCAAGGCTGGAGTAAAACTGGAAGTAGCCAACATGCAGGCTGAACTGGTGGCAAAACCCTCCGTGTCTGTGGAGTTTGTGACAAATATGGGCATCATCATTCCGGACTTCGCTAGGAGTGGGGTCCAGATGAACACCAACTTCTTCCACGAGTCGGGTCTGGAGGCTCATGTTGCCCTAAAAGCTGGGAAGCTGAAGTTTATCATTCCTTCCCCAAAGAGACCAGTCAAGCTGCTCAGTGGAGGCAACACATTACATTTGGTCTCTACCACCAAAACGGAGGTGATCCCACCTCTCATTGAGAACAGGCAGTCCTGGTCAGTTTGCAAGCAAGTCTTTCCTGGCCTGAATTACTGCACCTCAGGCGCTTACTCCAACGCCAGCTCCACAGACTCCGCCTCCTACTATCCGCTGACCGGGGACACCAGATTAGAGCTGGAACTGAGGCCTACAGGAGAGATTGAGCAGTATTCTGTCAGCGCAACCTATGAGCTCCAGAGAGAGGACAGAGCCTTGGTGGATACCCTGAAGTTTGTAACTCAAGCAGAAGGTGCGAAGCAGACTGAGGCTACCATGACATTCAAATATAATCGGCAGAGTATGACCTTGTCCAGTGAAGTCCAAATTCCGGATTTTGATGTTGACCTCGGAACAATCCTCAGAGTTAATGATGAATCTACTGAGGGCAAAACGTCTTACAGACTCACCCTGGACATTCAGAACAAGAAAATTACTGAGGTCGCCCTCATGGGCCACCTAAGTTGTGACACAAAGGAAGAAAGAAAAATCAAGGGTGTTATTTCCATACCCCGTTTGCAAGCAGAAGCCAGAAGTGAGATCCTCGCCCACTGGTCGCCTGCCAAACTGCTTCTCCAAATGGACTCATCTGCTACAGCTTATGGCTCCACAGTTTCCAAGAGGGTGGCATGGCATTATGATGAAGAGAAGATTGAATTTGAATGGAACACAGGCACCAATGTAGATACCAAAAAAATGACTTCCAATTTCCCTGTGGATCTCTCCGATTATCCTAAGAGCTTGCATATGTATGCTAATAGACTCCTGGATCACAGAGTCCCTGAAACAGACATGACTTTCCGGCACGTGGGTTCCAAATTAATAGTTGCAATGAGCTCATGGCTTCAGAAGGCATCTGGGAGTCTTCCTTATACCCAGACTTTGCAAGACCACCTCAATAGCCTGAAGGAGTTCAACCTCCAGAACATGGGATTGCCAGACTTCCACATCCCAGAAAACCTCTTCTTAAAAAGCGATGGCCGGGTCAAATATACCTTGAACAAGAACAGTTTGAAAATTGAGATTCCTTTGCCTTTTGGTGGCAAATCCTCCAGAGATCTAAAGATGTTAGAGACTGTTAGGACACCAGCCCTCCACTTCAAGTCTGTGGGATTCCATCTGCCATCTCGAGAGTTCCAAGTCCCTACTTTTACCATTCCCAAGTTGTATCAACTGCAAGTGCCTCTCCTGGGTGTTCTAGACCTCTCCACGAATGTCTACAGCAACTTGTACAACTGGTCCGCCTCCTACAGTGGTGGCAACACCAGCACAGACCATTTCAGCCTTCGGGCTCGTTACCACATGAAGGCTGACTCTGTGGTTGACCTGCTTTCCTACAATGTGCAAGGATCTGGAGAAACAACATATGACCACAAGAATACGTTCACACTATCATGTGATGGGTCTCTACGCCACAAATTTCTAGATTCGAATATCAAATTCAGTCATGTAGAAAAACTTGGAAACAACCCAGTCTCAAAAGGTTTACTAATATTCGATGCATCTAGTTCCTGGGGACCACAGATGTCTGCTTCAGTTCATTTGGACTCCAAAAAGAAACAGCATTTGTTTGTCAAAGAAGTCAAGATTGATGGGCAGTTCAGAGTCTCTTCGTTCTATGCTAAAGGCACATATGGCCTGTCTTGTCAGAGGGATCCTAACACTGGCCGGCTCAATGGAGAGTCCAACCTGAGGTTTAACTCCTCCTACCTCCAAGGCACCAACCAGATAACAGGAAGATATGAAGATGGAACCCTCTCCCTCACCTCCACCTCTGATCTGCAAAGTGGCATCATTAAAAATACTGCTTCCCTAAAGTATGAGAACTACGAGCTGACTTTAAAATCTGACACCAATGGGAAGTATAAGAACTTTGCCACTTCTAACAAGATGGATATGACCTTCTCTAAGCAAAATGCACTGCTGCGTTCTGAATATCAGGCTGATTACGAGTCATTGAGGTTCTTCAGCCTGCTTTCTGGATCACTAAATTCCCATGGTCTTGAGTTAAATGCTGACATCTTAGGCACTGACAAAATTAATAGTGGTGCTCACAAGGCGACACTAAGGATTGGCCAAGATGGAATATCTACCAGTGCAACGACCAACTTGAAGTGTAGTCTCCTGGTGCTGGAGAATGAGCTGAATGCAGAGCTTGGCCTCTCTGGGGCATCTATGAAATTAACAACAAATGGCCGCTTCAGGGAACACAATGCAAAATTCAGTCTGGATGGGAAAGCCGCCCTCACAGAGCTATCACTGGGAAGTGCTTATCAGGCCATGATTCTGGGTGTCGACAGCAAAAACATTTTCAACTTCAAGGTCAGTCAAGAAGGACTTAAGCTCTCAAATGACATGATGGGCTCATATGCTGAAATGAAATTTGACCACACAAACAGTCTGAACATTGCAGGCTTATCACTGGACTTCTCTTCAAAACTTGACAACATTTACAGCTCTGACAAGTTTTATAAGCAAACTGTTAATTTACAGCTACAGCCCTATTCTCTGGTAACTACTTTAAACAGTGACCTGAAATACAATGCTCTGGATCTCACCAACAATGGGAAACTACGGCTAGAACCCCTGAAGCTGCATGTGGCTGGTAACCTAAAAGGAGCCTACCAAAATAATGAAATAAAACACATCTATGCCATCTCTTCTGCTGCCTTATCAGCAAGCTATAAAGCAGACACTGTTGCTAAGGTTCAGGGTGTGGAGTTTAGCCATCGGCTCAACACAGACATCGCTGGGCTGGCTTCAGCCATTGACATGAGCACAAACTATAATTCAGACTCACTGCATTTCAGCAATGTCTTCCGTTCTGTAATGGCCCCGTTTACCATGACCATCGATGCACATACAAATGGCAATGGGAAACTCGCTCTCTGGGGAGAACATACTGGGCAGCTGTATAGCAAATTCCTGTTGAAAGCAGAACCTCTGGCATTTACTTTCTCTCATGATTACAAAGGCTCCACAAGTCATCATCTCGTGTCTAGGAAAAGCATCAGTGCAGCTCTTGAACACAAAGTCAGTGCCCTGCTTACTCCAGCTGAGCAGACAGGCACCTGGAAACTCAAGACCCAATTTAACAACAATGAATACAGCCAGGACTTGGATGCTTACAACACTAAAGATAAAATTGGCGTGGAGCTTACTGGACGAACTCTGGCTGACCTAACTCTACTAGACTCCCCAATTAAAGTGCCACTTTTACTCAGTGAGCCCATCAATATCATTGATGCTTTAGAGATGAGAGATGCCGTTGAGAAGCCCCAAGAATTTACAATTGTTGCTTTTGTAAAGTATGATAAAAACCAAGATGTTCACTCCATTAACCTCCCATTTTTTGAGACCTTGCAAGAATATTTTGAGAGGAATCGACAAACCATTATAGTTGTAGTGGAAAACGTACAGAGAAACCTGAAGCACATCAATATTGATCAATTTGTAAGAAAATACAGAGCAGCCCTGGGAAAACTCCCACAGCAAGCTAATGATTATCTGAATTCATTCAATTGGGAGAGACAAGTTTCACATGCCAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAATTACAGAAAATGATATACAAATTGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTGCAGACATATATGATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTATTGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTGATGAGCACTATCATATCCGTGTAAATTTAGTAAAAACAATCCATGATCTACATTTGTTTATTGAAAATATTGATTTTAACAAAAGTGGAAGTAGTACTGCATCCTGGATTCAAAATGTGGATACTAAGTACCAAATCAGAATCCAGATACAAGAAAAACTGCAGCAGCTTAAGAGACACATACAGAATATAGACATCCAGCACCTAGCTGGAAAGTTAAAACAACACATTGAGGCTATTGATGTTAGAGTGCTTTTAGATCAATTGGGAACTACAATTTCATTTGAAAGAATAAATGATGTTCTTGAGCATGTCAAACACTTTGTTATAAATCTTATTGGGGATTTTGAAGTAGCTGAGAAAATCAATGCCTTCAGAGCCAAAGTCCATGAGTTAATCGAGAGGTATGAAGTAGACCAACAAATCCAGGTTTTAATGGATAAATTAGTAGAGTTGACCCACCAATACAAGTTGAAGGAGACTATTCAGAAGCTAAGCAATGTCCTACAACAAGTTAAGATAAAAGATTACTTTGAGAAATTGGTTGGATTTATTGATGATGCTGTGAAGAAGCTTAATGAATTATCTTTTAAAACATTCATTGAAGATGTTAACAAATTCCTTGACATGTTGATAAAGAAATTAAAGTCATTTGATTACCACCAGTTTGTAGATGAAACCAATGACAAAATCCGTGAGGTGACTCAGAGACTCAATGGTGAAATTCAGGCTCTGGAACTACCACAAAAAGCTGAAGCATTAAAACTGTTTTTAGAGGAAACCAAGGCCACAGTTGCAGTGTATCTGGAAAGCCTACAGGACACCAAAATAACCTTAATCATCAATTGGTTACAGGAGGCTTTAAGTTCAGCATCTTTGGCTCACATGAAGGCCAAATTCCGAGAGACTCTAGAAGATACACGAGACCGAATGTATCAAATGGACATTCAGCAGGAACTTCAACGATACCTGTCTCTGGTAGGCCAGGTTTATAGCACACTTGTCACCTACATTTCTGATTGGTGGACTCTTGCTGCTAAGAACCTTACTGACTTTGCAGAGCAATATTCTATCCAAGATTGGGCTAAACGTATGAAAGCATTGGTAGAGCAAGGGTTCACTGTTCCTGAAATCAAGACCATCCTTGGGACCATGCCTGCCTTTGAAGTCAGTCTTCAGGCTCTTCAGAAAGCTACCTTCCAGACACCTGATTTTATAGTCCCCCTAACAGATTTGAGGATTCCATCAGTTCAGATAAACTTCAAAGACTTAAAAAATATAAAAATCCCATCCAGGTTTTCCACACCAGAATTTACCATCCTTAACACCTTCCACATTCCTTCCTTTACAATTGACTTTGTCGAAATGAAAGTAAAGATCATCAGAACCATTGACCAGATGCAGAACAGTGAGCTGCAGTGGCCCGTTCCAGATATATATCTCAGGGATCTGAAGGTGGAGGACATTCCTCTAGCGAGAATCACCCTGCCAGACTTCCGTTTACCAGAAATCGCAATTCCAGAATTCATAATCCCAACTCTCAACCTTAATGATTTTCAAGTTCCTGACCTTCACATACCAGAATTCCAGCTTCCCCACATCTCACACACAATTGAAGTACCTACTTTTGGCAAGCTATACAGTATTCTGAAAATCCAATCTCCTCTTTTCACATTAGATGCAAATGCTGACATAGGGAATGGAACCACCTCAGCAAACGAAGCAGGTATCGCAGCTTCCATCACTGCCAAAGGAGAGTCCAAATTAGAAGTTCTCAATTTTGATTTTCAAGCAAATGCACAACTCTCAAACCCTAAGATTAATCCGCTGGCTCTGAAGGAGTCAGTGAAGTTCTCCAGCAAGTACCTGAGAACGGAGCATGGGAGTGAAATGCTGTTTTTTGGAAATGCTATTGAGGGAAAATCAAACACAGTGGCAAGTTTACACACAGAAAAAAATACACTGGAGCTTAGTAATGGAGTGATTGTCAAGATAAACAATCAGCTTACCCTGGATAGCAACACTAAATACTTCCACAAATTGAACATCCCCAAACTGGACTTCTCTAGTCAGGCTGACCTGCGCAACGAGATCAAGACACTGTTGAAAGCTGGCCACATAGCATGGACTTCTTCTGGAAAAGGGTCATGGAAATGGGCCTGCCCCAGATTCTCAGATGAGGGAACACATGAATCACAAATTAGTTTCACCATAGAAGGACCCCTCACTTCCTTTGGACTGTCCAATAAGATCAATAGCAAACACCTAAGAGTAAACCAAAACTTGGTTTATGAATCTGGCTCCCTCAACTTTTCTAAACTTGAAATTCAATCACAAGTCGATTCCCAGCATGTGGGCCACAGTGTTCTAACTGCTAAAGGCATGGCACTGTTTGGAGAAGGGAAGGCAGAGTTTACTGGGAGGCATGATGCTCATTTAAATGGAAAGGTTATTGGAACTTTGAAAAATTCTCTTTTCTTTTCAGCCCAGCCATTTGAGATCACGGCATCCACAAACAATGAAGGGAATTTGAAAGTTCGTTTTCCATTAAGGTTAACAGGGAAGATAGACTTCCTGAATAACTATGCACTGTTTCTGAGTCCCAGTGCCCAGCAAGCAAGTTGGCAAGTAAGTGCTAGGTTCAATCAGTATAAGTACAACCAAAATTTCTCTGCTGGAAACAACGAGAACATTATGGAGGCCCATGTAGGAATAAATGGAGAAGCAAATCTGGATTTCTTAAACATTCCTTTAACAATTCCTGAAATGCGTCTACCTTACACAATAATCACAACTCCTCCACTGAAAGATTTCTCTCTATGGGAAAAAACAGGCTTGAAGGAATTCTTGAAAACGACAAAGCAATCATTTGATTTAAGTGTAAAAGCTCAGTATAAGAAAAACAAACACAGGCATTCCATCACAAATCCTTTGGCTGTGCTTTGTGAGTTTATCAGTCAGAGCATCAAATCCTTTGACAGGCATTTTGAAAAAAACAGAAACAATGCATTAGATTTTGTCACCAAATCCTATAATGAAACAAAAATTAAGTTTGATAAGTACAAAGCTGAAAAATCTCACGACGAGCTCCCCAGGACCTTTCAAATTCCTGGATACACTGTTCCAGTTGTCAATGTTGAAGTGTCTCCATTCACCATAGAGATGTCGGCATTCGGCTATGTGTTCCCAAAAGCAGTCAGCATGCCTAGTTTCTCCATCCTAGGTTCTGACGTCCGTGTGCCTTCATACACATTAATCCTGCCATCATTAGAGCTGCCAGTCCTTCATGTCCCTAGAAATCTCAAGCTTTCTCTTCCACATTTCAAGGAATTGTGTACCATAAGCCATATTTTTATTCCTGCCATGGGCAATATTACCTATGATTTCTCCTTTAAATCAAGTGTCATCACACTGAATACCAATGCTGAACTTTTTAACCAGTCAGATATTGTTGCTCATCTCCTTTCTTCATCTTCATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGACAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTCATAACAGTACTGTGAGCTTAACCACGAAAAATATGGAAGTGTCAGTGGCAAAAACCACAAAAGCCGAAATTCCAATTTTGAGAATGAATTTCAAGCAAGAACTTAATGGAAATACCAAGTCAAAACCTACTGTCTCTTCCTCCATGGAATTTAAGTATGATTTCAATTCTTCAATGCTGTACTCTACCGCTAAAGGAGCAGTTGACCACAAGCTTAGCTTGGAAAGCCTCACCTCTTACTTTTCCATTGAGTCATCTACCAAAGGAGATGTCAAGGGTTCGGTTCTTTCTCGGGAATATTCAGGAACTATTGCTAGTGAGGCCAACACTTACTTGAATTCCAAGAGCACACGGTCTTCAGTGAAGCTGCAGGGCACTTCCAAAATTGATGATATCTGGAACCTTGAAGTAAAAGAAAATTTTGCTGGAGAAGCCACACTCCAACGCATATATTCCCTCTGGGAGCACAGTACGAAAAACCACTTACAGCTAGAGGGCCTCTTTTTCACCAACGGAGAACATACAAGCAAAGCCACCCTGGAACTCTCTCCATGGCAAATGTCAGCTCTTGTTCAGGTCCATGCAAGTCAGCCCAGTTCCTTCCATGATTTCCCTGACCTTGGCCAGGAAGTGGCCCTGAATGCTAACACTAAGAACCAGAAGATCAGATGGAAAAATGAAGTCCGGATTCATTCTGGGTCTTTCCAGAGCCAGGTCGAGCTTTCCAATGACCAAGAAAAGGCACACCTTGACATTGCAGGATCCTTAGAAGGACACCTAAGGTTCCTCAAAAATATCATCCTACCAGTCTATGACAAGAGCTTATGGGATTTCCTAAAGCTGGATGTAACCACCAGCATTGGTAGGAGACAGCATCTTCGTGTTTCAACTGCCTTTGTGTACACCAAAAACCCCAATGGCTATTCATTCTCCATCCCTGTAAAAGTTTTGGCTGATAAATTCATTACTCCTGGGCTGAAACTAAATGATCTAAATTCAGTTCTTGTCATGCCTACGTTCCATGTCCCATTTACAGATCTTCAGGTTCCATCGTGCAAACTTGACTTCAGAGAAATACAAATCTATAAGAAGCTGAGAACTTCATCATTTGCCCTCAACCTACCAACACTCCCCGAGGTAAAATTCCCTGAAGTTGATGTGTTAACAAAATATTCTCAACCAGAAGACTCCTTGATTCCCTTTTTTGAGATAACCGTGCCTGAATCTCAGTTAACTGTGTCCCAGTTCACGCTTCCAAAAAGTGTTTCAGATGGCATTGCTGCTTTGGATCTAAATGCAGTAGCCAACAAGATCGCAGACTTTGAGTTGCCCACCATCATCGTGCCTGAGCAGACCATTGAGATTCCCTCCATTAAGTTCTCTGTACCTGCTGGAATTGTCATTCCTTCCTTTCAAGCACTGACTGCACGCTTTGAGGTAGACTCTCCCGTGTATAATGCCACTTGGAGTGCCAGTTTGAAAAACAAAGCAGATTATGTTGAAACAGTCCTGGATTCCACATGCAGCTCAACCGTACAGTTCCTAGAATATGAACTAAATGTTTTGGGAACACACAAAATCGAAGATGGTACGTTAGCCTCTAAGACTAAAGGAACACTTGCACACCGTGACTTCAGTGCAGAATATGAAGAAGATGGCAAATTTGAAGGACTTCAGGAATGGGAAGGAAAAGCGCACCTCAATATCAAAAGCCCAGCGTTCACCGATCTCCATCTGCGCTACCAGAAAGACAAGAAAGGCATCTCCACCTCAGCAGCCTCCCCAGCCGTAGGCACCGTGGGCATGGATATGGATGAAGATGACGACTTTTCTAAATGGAACTTCTACTACAGCCCTCAGTCCTCTCCAGATAAAAAACTCACCATATTCAAAACTGAGTTGAGGGTCCGGGAATCTGATGAGGAAACTCAGATCAAAGTTAATTGGGAAGAAGAGGCAGCTTCTGGCTTGCTAACCTCTCTGAAAGACAACGTGCCCAAGGCCACAGGGGTCCTTTATGATTATGTCAACAAGTACCACTGGGAACACACAGGGCTCACCCTGAGAGAAGTGTCTTCAAAGCTGAGAAGAAATCTGCAGAACAATGCTGAGTGGGTTTATCAAGGGGCCATTAGGCAAATTGATGATATCGACGTGAGGTTCCAGAAAGCAGCCAGTGGCACCACTGGGACCTACCAAGAGTGGAAGGACAAGGCCCAGAATCTGTACCAGGAACTGTTGACTCAGGAAGGCCAAGCCAGTTTCCAGGGACTCAAGGATAACGTGTTTGATGGCTTGGTACGAGTTACTCAAAAATTCCATATGAAAGTCAAGCATCTGATTGACTCACTCATTGATTTTCTGAACTTCCCCAGATTCCAGTTTCCGGGGAAACCTGGGATATACACTAGGGAGGAACTTTGCACTATGTTCATAAGGGAGGTAGGGACGGTACTGTCCCAGGTATATTCGAAAGTCCATAATGGTTCAGAAATACTGTTTTCCTATTTCCAAGACCTAGTGATTACACTTCCTTTCGAGTTAAGGAAACATAAACTAATAGATGTAATCTCGATGTATAGGGAACTGTTGAAAGATTTATCAAAAGAAGCCCAAGAGGTATTTAAAGCCATTCAGTCTCTCAAGACCACAGAGGTGCTACGTAATCTTCAGGACCTTTTACAATTCATTTTCCAACTAATAGAAGATAACATTAAACAGCTGAAAGAGATGAAATTTACTTATCTTATTAATTATATCCAAGATGAGATCAACACAATCTTCAATGATTATATCCCATATGTTTTTAAATTGTTGAAAGAAAACCTATGCCTTAATCTTCATAAGTTCAATGAATTTATTCAAAACGAGCTTCAGGAAGCTTCTCAAGAGTTACAGCAGATCCATCAATACATTATGGCCCTTCGTGAAGAATATTTTGATCCAAGTATAGTTGGCTGGACAGTGAAATATTATGAACTTGAAGAAAAGATAGTCAGTCTGATCAAGAACCTGTTAGTTGCTCTTAAGGACTTCCATTCTGAATATATTGTCAGTGCCTCTAACTTTACTTCCCAACTCTCAAGTCAAGTTGAGCAATTTCTGCACAGAAATATTCAGGAATATCTTAGCATCCTTACCGATCCAGATGGAAAAGGGAAAGAGAAGATTGCAGAGCTTTCTGCCACTGCTCAGGAAATAATTAAAAGCCAGGCCATTGCGACGAAGAAAATAATTTCTGATTACCACCAGCAGTTTAGATATAAACTGCAAGATTTTTCAGACCAACTCTCTGATTACTATGAAAAATTTATTGCTGAATCCAAAAGATTGATTGACCTGTCCATTCAAAACTACCACACATTTCTGATATACATCACGGAGTTACTGAAAAAGCTGCAATCAACCACAGTCATGAACCCCTACATGAAGCTTGCTCCAGGAGAACTTACTATCATCCTCTAATTTTTTAAAAGAAATCTTCATTTATTCTTCTTTTCCAATTGAACTTTCACATAGCACAGAAAAAATTCAAACTGCCTATATTGATAAAACCATACAGTGAGCCAGCCTTGCAGTAGGCAGTAGACTATAAGCAGAAGCACATATGAACTGGACCTGCACCAAAGCTGGCACCAGGGCTCGGAAGGTCTCTGAACTCAGAAGGATGGCATTTTTTGCAAGTTAAAGAAAATCAGGATCTGAGTTATTTTGCTAAACTTGGGGGAGGAGGAACAAATAAATGGAGTCTTTATTGTGTATCATA (SEQ ID NO: 6681)>gi|4557442|ref|NM_000078.1| Homo sapiens cholesteryl ester transferprotein, plasma (CETP), mRNAGTGAATCTCTGGGGCCAGGAAGACCCTGCTGCCCGGAAGAGCCTCATGTTCCGTGGGGGCTGGGCGGACATACATATACGGGCTCCAGGCTGAACGGCTCGGGCCACTTACACACCACTGCCTGATAACCATGCTGGCTGCCACAGTCCTGACCCTGGCCCTGCTGGGCAATGCCCATGCCTGCTCCAAAGGCACCTCGCACGAGGCAGGCATCGTGTGCCGCATCACCAAGCCTGCCCTCCTGGTGTTGAACCACGAGACTGCCAAGGTGATCCAGACCGCCTTCCAGCGAGCCAGCTACCCAGATATCACGGGCGAGAAGGCCATGATGCTCCTTGGCCAAGTCAAGTATGGGTTGCACAACATCCAGATCAGCCACTTGTCCATCGCCAGCAGCCAGGTGGAGCTGGTGGAAGCCAAGTCCATTGATGTCTCCATTCAGAACGTGTCTGTGGTCTTCAAGGGGACCCTGAAGTATGGCTACACCACTGCCTGGTGGCTGGGTATTGATCAGTCCATTGACTTCGAGATCGACTCTGCCATTGACCTCCAGATCAACACACAGCTGACCTGTGACTCTGGTAGAGTGCGGACCGATGCCCCTGACTGCTACCTGTCTTTCCATAAGCTGCTCCTGCATCTCCAAGGGGAGCGAGAGCCTGGGTGGATCAAGCAGCTGTTCACAAATTTCATCTCCTTCACCCTGAAGCTGGTCCTGAAGGGACAGATCTGCAAAGAGATCAACGTCATCTCTAACATCATGGCCGATTTTGTCCAGACAAGGGCTGCCAGCATCCTTTCAGATGGAGACATTGGGGTGGACATTTCCCTGACAGGTGATCCCGTCATCACAGCCTCCTACCTGGAGTCCCATCACAAGGGTCATTTCATCTACAAGAATGTCTCAGAGGACCTCCCCCTCCCCACCTTCTCGCCCACACTGCTGGGGGACTCCCGCATGCTGTACTTCTGGTTCTCTGAGCGAGTCTTCCACTCGCTGGCCAAGGTAGCTTTCCAGGATGGCCGCCTCATGCTCAGCCTGATGGGAGACGAGTTCAAGGCAGTGCTGGAGACCTGGGGCTTCAACACCAACCAGGAAATCTTCCAAGAGGTTGTCGGCGGCTTCCCCAGCCAGGCCCAAGTCACCGTCCACTGCCTCAAGATGCCCAAGATCTCCTGCCAAAACAAGGGAGTCGTGGTCAATTCTTCAGTGATGGTGAAATTCCTCTTTCCACGCCCAGACCAGCAACATTCTGTAGCTTACACATTTGAAGAGGATATCGTGACTACCGTCCAGGCCTCCTATTCTAAGAAAAAGCTCTTCTTAAGCCTCTTGGATTTCCAGATTACACCAAAGACTGTTTCCAACTTGACTGAGAGCAGCTCCGAGTCCATCCAGAGCTTCCTGCAGTCAATGATCACCGCTGTGGGCATCCCTGAGGTCATGTCTCGGCTCGAGGTAGTGTTTACAGCCCTCATGAACAGCAAAGGCGTGAGCCTCTTCGACATCATCAACCCTGAGATTATCACTCGAGATGGCTTCCTGCTGCTGCAGATGGACTTTGGCTTCCCTGAGCACCTGCTGGTGGATTTCCTCCAGAGCTTGAGCTAGAAGTCTCCAAGGAGGTCGGGATGGGGCTTGTAGCAGAAGGCAAGCACCAGGCTCACAGCTGGAACCCTGGTGTCTCCTCCAGCGTGGTGGAAGTTGGGTTAGGAGTACGGAGATGGAGATTGGCTCCCAACTCCTCCCTATCCTAAAGGCCCACTGGCATTAAAGTGCTGTATCCAAG (SEQ ID NO:6682) >gi|414668|emb|X75500.1|HSMTP H. sapiens mRNA for microsomaltriglyceride transfer proteinTGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATTCAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTCCACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAACGTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGGATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAAAATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTCAAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTATTTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCAGGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACGACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCTTTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAAAATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAGGCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGAGCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAACCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAAGAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTGCTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCTCCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATTAGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTGTCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGGAGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTTCCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCACTGCTCTCCAGAGATATGATCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACCACCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCCATCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATGCTCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGGAAATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAACGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGAAGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCCAAGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATGTCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCCAAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTCAGGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTCAGGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTAATAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGCAGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAAGGATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCTCAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCAAAGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTACTTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGTTTACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTATATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTACTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGTTAAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGTATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAAAAAAAAAAAAAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAGAAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGTTAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATC (SEQ ID NO:6683) >gi|1217638|emb|X91148.1|HSMTTP H. sapiens mRNA for microsomaltriglyceride transfer proteinTGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATTCAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTCCACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAACGTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGGATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAAAATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTCAAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTATTTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCAGGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACGACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCTTTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAAAATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAGGCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGAGCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAACCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAAGAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTGCTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCTCCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATTAGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTGTCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGGAGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTTCCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCACTGCTCTCCAGAGATATGATGCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACCACCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCCATCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATGCTCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGGAAATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAACGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGAAGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCCAAGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATGTCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCCAAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTCAGGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTCAGGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTAATAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGCAGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAAGGATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCTCAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCAAAGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTACTTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGTTTACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTATATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTACTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGTTAAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGTATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAAAAAAAAAAAAGAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAGAAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGTTAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATCTCATCTTTCTGCTGACCATTTTAGTGAGGTTGTTCCAAAGAGCATTCAGGTCTCTACCTCCAGCCCTGCAAAAATATTGGACCTAGCACAGAGGAATCAGGAAAATTAATTTCAGAAACTCCATTTGATTTTTCTTTTGCTGTGTCTTTTTTGAGACTGTAATATGGTACACTGTCCTCTAAGGACATCCTCATTTTATCTCACCTTTTTGGGGGTGAGAGCTCTAGTTCATTTAACTGTACTCTGCACAATAGCTAGGATGACTAAGAGAACATTGCTTCAAGAAACTGGTGGATTTGGATTTCCAAAATATGAAATAAGGAGAAAAATGTTTTTATTTGTATGAATTAAAAGATCCATGTTGAACATTTGCAAATATTTATTAATAAACAGATGTGGTGATAAACCCAAAACAAATGACAGGTGCTTATTTTCCACTAAACACAGACACATGAAATGAAAGTTTAGCTAGCCCACTATTTGTTGTAAATTGAAAACGAAGTGTGATAAAATAAATATGTAGAAATCAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6684)>gi|21361125|ref|NM_001467.2| Homo sapiens glucose-6-phosphatase,transport (glucose-6-phosphate) protein 1 (G6PT1), mRNAGGCACGAGGGGCCACCGAGGCGCTGTCCCTGACCACCAGCACGAGACCCCTTTCTATCGCGCCAGTCCTGTGGTCTCCGCACCTCTCCAGCTCCTGCACCCCCGGCCCCCGTGGTTCCCAGCCGCACAGTAGCGTGTCCTGGGTAGCGTGAGGACCCACGGGGCTGAGCAGGTGCCACGAGCCCGCCGCCTCTTCGCCGCCCGCCGCCTCTCCTCCTCTCCCGCCCGCCGCCTGGCCCTCCCCTACCAGGCTGAGCCTCTGGCTGCCAGAAGCGCGGGGCCTCCGGGAGAATACGTGCGGTCGCCCGCTCCGCGTGCGCCTACGCCTTCTGCTCCAGTTGCTTTCCCAATTGAGCGGAAAAGCCGGGGCATGTTGCCGGGGCCCTGGGCGGGACGGTTGTGCCCTGCAGCCCGAAGCCCGCCGGGGCACCTTCCCGCCCACGAGCTGCCCAGTCCCTCTGCTTGCGGCCCCTGCCAACGTCCCACAGGACACTGGGTCCCCTTGGAGCCTCCCCAGGCTTAATGATTGTCCAGAAGGCGGCTATAAAGGGAGCCTGGGAGGCTGGGTGGAGGAGGGAGCAGAAAAAACCCAACTCAGCAGATCTGGGAACTGTGAGAGCGGCAAGCAGGAACTGTGGTCAGAGGCTGTGCGTCTTGGCTGGTAGGGCCTGCTCTTTTCTACCATGGCAGCCCAGGGCTATGGCTATTATCGCACTGTGATCTTCTCAGCCATGTTTGGGGGCTACAGCCTGTATTACTTCAATCGCAAGACCTTCTCCTTTGTCATGCCATCATTGGTGGAAGAGATCCCTTTGGACAAGGATGATTTGGGGTTCATCACCAGCAGCCAGTCGGCAGCTTATGCTATCAGCAAGTTTGTCAGTGGGGTGCTGTCTGACCAGATGAGTGCTCGCTGGCTCTTCTCTTCTGGGCTGCTCCTGGTTGGCCTGGTCAACATATTCTTTGCCTGGAGCTCCACAGTACCTGTCTTTGCTGCCCTCTGGTTCCTTAATGGCCTGGCCCAGGGGCTGGGCTGGCCCCCATGTGGGAAGGTCCTGCGGAAGTGGTTTGAGCCATCTCAGTTTGGCACTTGGTGGGCCATCCTGTCAACCAGCATGAACCTGGCTGGAGGGCTGGGCCCTATCCTGGCAACCATCCTTGCCCAGAGCTACAGCTGGCGCAGCACGCTGGCCCTATCTGGGGCACTGTGTGTGGTTGTCTCCTTCCTCTGTCTCCTGCTCATCCACAATGAACCTGCTGATGTTGGACTCCGCAACCTGGACCCCATGCCCTCTGAGGGCAAGAAGGGCTCCTTGAAGGAGGAGAGCACCCTGCAGGAGCTGCTGCTGTCCCCTTACCTGTGGGTGCTCTCCACTGGTTACCTTGTGGTGTTTGGAGTAAAGACCTGCTGTACTGACTGGGGCCAGTTCTTCCTTATCCAGGAGAAAGGACAGTCAGCCCTTGTAGGTAGCTCCTACATGAGTGCCCTGGAAGTTGGGGGCCTTGTAGGCAGCATCGCAGCTGGCTACCTGTCAGACCGGGCCATGGCAAAGGCGGGACTGTCCAACTACGGGAACCCTCGCCATGGCCTGTTGCTGTTCATGATGGCTGGCATGACAGTGTCCATGTACCTCTTCCGGGTAACAGTGACCAGTGACTCCCCCAAGCTCTGGATCCTGGTATTGGGAGCTGTATTTGGTTTCTCCTCGTATGGCCCCATTGCCCTGTTTGGAGTCATAGCCAACGAGAGTGCCCCTCCCAACTTGTGTGGCACCTCCCACGCCATTGTGGGACTCATGGCCAATGTGGGCGGCTTTCTGGCTGGGCTGCCCTTCAGCACCATTGCCAAGCACTACAGTTGGAGCACAGCCTTCTGGGTGGCTGAAGTGATTTGTGCGGCCAGCACGGCTGCCTTCTTCCTCCTACGAAACATCCGCACCAAGATGGGCCGAGTGTCCAAGAAGGCTGAGTGAAGAGAGTCCAGGTTCCGGAGCACCATCCCACGGTGGCCTTCCCCCTGCACGCTCTGCGGGGAGAAAAGGAGGGGCCTGCCTGGCTAGCCCTGAACCTTTCACTTTCCATTTCTGCGCCTTTTCTGTCACCCGGGTGGCGCTGGAAGTTATCAGTGGCTAGTGAGGTCCCAGCTCCCTGATCCTATGCTCTATTTAAAAGATAACCTTTGGCCTTAGACTCCGTTAGCTCCTATTTCCTGCCTTCAGACAAACAGGAAACTTCTGCAGTCAGGAAGGCTCCTGTACCCTTCTTCTTTTCCTAGGCCCTGTCCTGCCCGCATCCTACCCCATCCCCACCTGAAGTGAGGCTATCCCTGCAGCTGCAGGGCACTAATGACCCTTGACTTCTGCTGGGTCCTAAGTCCTCTCAGCAGTGGGTGACTGCTGTTGCCAATACCTCAGACTCCAGGGAAAGAGAGGAGGCCATCATTCTCACTGTACCACTAGGCGCAGTTGGATATAGGTGGGAAGAAAAGGTGACTTGTTATAGAAGATTAAAACTAGATTTGATACTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6685)gi|4503130|ref|NM_001904.1| Homo sapiens catenin (cadherin-associatedprotein), beta 1, 88 kDa (CTNNB1), mRNAAAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGGAGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTCGGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAGAGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGATCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTTGATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGACACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCTGCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGGAAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAAAGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGGATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGAAGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAGTTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGTGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGCTGTATTGTCTGAACTTGCATTGTGATTGGCCTGTAGAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGACACACTAACCAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGAGGAGTAACAATACAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGATGGAATTTAGCAAACCCTAGCCTTGCTTGTTAAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATGGTACTGACTTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTTTTTAAGTCTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGTAATGGTGTAGAACACTAATTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTGTGTAGCCTTTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAATAAGCAGGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAGTAAGAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAACCTGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAATTCATT (SEQ ID NO: 6686) gi|18104977|ref|NM_002827.2| Homo sapiens proteintyrosine phosphatase, non-receptor type 1 (PTPN1), mRNAGTGATGCGTAGTTCCGGCTGCCGGTTGACATGAAGAAGCAGCAGCGGCTAGGGCGGCGGTAGCTGCAGGGGTCGGGGATTGCAGCGGGCCTCGGGGCTAAGAGCGCGACGCGGCCTAGAGCGGCAGACGGCGCAGTGGGCCGAGAAGGAGGCGCAGCAGCCGCCCTGGCCCGTCATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAGTCCGGGAGCTGGGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCAAGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTCGGATTAAACTACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGGAGTTACATTCTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGGGTGTCGTCATGCTCAACAGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGAAGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATCAAGTCATATTATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCCACTATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCGAGAGTCAGGGTCACTCAGCCCGGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCTGGAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGATATCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTTCTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAGGAGCTTTCCCACGAGGACCTGGAGCCCCCACCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAATCCTGGAGCCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTGAAGGAAGAGACCCAGGAGGATAAAGACTGCCCCATCAAGGAAGAAAAAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAAAGCATGAGTCAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGGTGCCCAGGCTGCCTCCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGAAGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTTCCTGGTCAACATGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGTTCCTGTTCAACAGCAACACATAGCCTGACCCTCCTCCACTCCACCTCCACCCACTGTCCGCCTCTGCCCGCAGAGCCCACGCCCGACTAGCAGGCATGCCGCGGTAGGTAAGGGCCGCCGGACCGCGTAGAGAGCCGGGCCCCGGACGGACGTTGGTTCTGCACTAAAACCCATCTTCCCCGGATGTGTGTCTCACCCCTCATCCTTTTACTTTTTGCCCCTTCCACTTTGAGTACCAAATCCACAAGCCATTTTTTGAGGAGAGTGAAAGAGAGTACCATGCTGGCGGCGCAGAGGGAAGGGGCCTACACCCGTCTTGGGGCTCGCCCCACCCAGGGCTCCCTCCTGGAGCATCCCAGGCGGGCGGCACGCCAACAGCCCCCCCCTTGAATCTGCAGGGAGCAACTCTCCACTCCATATTTATTTAAACAATTTTTTCCCCAAAGGCATCCATAGTGCACTAGCATTTTCTTGAACCAATAATGTATTAAAATTTTTTGATGTCAGCCTTGCATCAAGGGCTTTATCAAAAAGTACAATAATAAATCCTCAGGTAGTACTGGGAATGGAAGGCTTTGCCATGGGCCTGCTGCGTCAGACCAGTACTGGGAAGGAGGACGGTTGTAAGCAGTTGTTATTTAGTGATATTGTGGGTAACGTGAGAAGATAGAACAATGCTATAATATATAATGAACACGTGGGTATTTAATAAGAAACATGATGTGAGATTACTTTGTCCCGCTTATTCTCCTCCCTGTTATCTGCTAGATCTAGTTCTCAATCACTGCTCCCCCGTGTGTATTAGAATGCATGTAAGGTCTTCTTGTGTCCTGATGAAAAATATGTGCTTGAAATGAGAAACTTTGATCTCTGCTTACTAATGTGCCCCATGTCCAAGTCCAACCTGCCTGTGCATGACCTGATCATTACATGGCTGTGGTTCCTAAGCCTGTTGCTGAAGTCATTGTCGCTCAGCAATAGGGTGCAGTTTTCCAGGAATAGGCATTTGCCTAATTCCTGGCATGACACTCTAGTGACTTCCTGGTGAGGCCCAGCCTGTCCTGGTACAGCAGGGTCTTGCTGTAACTCAGACATTCCAAGGGTATGGGAAGCCATATTCACACCTCACGCTCTGGACATGATTTAGGGAAGCAGGGACACCCCCCGCCCCCCACCTTTGGGATCAGCCTCCGCCATTCCAAGTCAACACTCTTCTTGAGCAGACCGTGATTTGGAAGAGAGGCACCTGCTGGAAACCACACTTCTTGAAACAGCCTGGGTGACGGTCCTTTAGGCAGCCTGCCGCCGTCTCTGTCCCGGTTCACCTTGCCGAGAGAGGCGCGTCTGCCCCACCCTCAAACCCTGTGGGGCCTGATGGTGCTCACGACTCTTCCTGCAAAGGGAACTGAAGACCTCCACATTAAGTGGCTTTTTAACATGAAAAACACGGCAGCTGTAGCTCCCGAGCTACTCTCTTGCCAGCATTTTCACATTTTGCCTTTCTCGTGGTAGAAGCCAGTACAGAGAAATTCTGTGGTGGGAACATTCGAGGTGTCACCCTGCAGAGCTATGGTGAGGTGTGGATAAGGCTTAGGTGCCAGGCTGTAAGCATTCTGAGCTGGGCTTGTTGTTTTTAAGTCCTGTATATGTATGTAGTAGTTTGGGTGTGTATATATAGTAGCATTTCAAAATGGACGTACTGGTTTAACCTCCTATCCTTGGAGAGCAGCTGGCTCTCCACCTTGTTACACATTATGTTAGAGAGGTAGCGAGCTGCTCTGCTATATGCCTTAAGCCAATATTTACTCATCAGGTCATTATTTTTTACAATGGCCATGGAATAAACCATTTTTACAAAA (SEQ ID NO: 6687)gi|12831192|gb|AF333324.1|Hepatitis C virus type 1b polyprotein mRNA,complete cdsGCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCACAAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTTAAGTTCCCGGGCGGTGGTCAGATCGTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGGCCCGAGGGTAGGACCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGTATGGGGTGGGCAGGATGGCTCCTGTCACCCCGTGGCTCTCGGCCTAGTTGGGGCCCCACAGACCCCCGGCGTAGGTCGCGTAATTTGGGTAAGGTCATCGATACCCTTACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTTGTCGGCGCCCCCCTAGGAGGCGCTGCCAGGGCCCTGGCGCATGGCGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAGGGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTAGCTTTGCTGTCTTGTTTGACCATCCCAGCTTCCGCTTACGAGGTGCGCAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGTATTGTGTATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGCGTGCCCTGCGTCCGGGAGAGTAATTTCTCCCGTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACAGCAGCATCCCCACCACGACAATACGACGCCACGTCGATTTGCTCGTTGGGGCGGCTGCTCTCTGTTCCGCTATGTACGTTGGGGATCTCTGCGGATCCGTTTTTCTCGTCTCCCAGCTGTTCACCTTCTCACCTCGCCGGTATGAGACGGTACAAGATTGCAATTGCTCAATCTATCCCGGCCACGTATCAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCACCTACAACGGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCCGTCGTGGACATGGTGGCGGGGGCCCACTGGGGTGTCCTAGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTGATTGTGATGCTACTCTTTGCTGGCGTTGACGGGCACACCCACGTGACAGGGGGAAGGGTAGCCTCCAGCACCCAGAGCCTCGTGTCCTGGCTCTCACAAGGGCCATCTCAGAAAATCCAACTCGTGAACACCAACGGCAGCTGGCACATCAACAGGACCGCTCTGAATTGCAATGACTCCCTCCAAACTGGGTTCATTGCTGCGCTGTTCTACGCACACAGGTTCAACGCGTCCGGATGTCCAGAGCGCATGGCCAGCTGCCGCCCCATCGACAAGTTCGCTCAGGGGTGGGGTCCCATCACTCACGTTGTGCCTAACATCTCGGACCAGAGGCCTTATTGCTGGCACTATGCACCCCAACCGTGCGGTATTGTACCCGCGTCGCAGGTGTGTGGCCCAGTGTATTGCTTCACCCCGAGTCCTGTTGTGGTGGGGACGACCGACCGTTCCGGAGTCCCCACGTATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTACTCAACAACACGCGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATAGCACCGGGTTCACCAAGACGTGCGGGGGCCCCCCGTGTAACATCGGGGGGGTTGGCAACAACACCTTGATTTGCCCCACGGATTGCTTCCGAAAGCACCCCGAGGCCACTTACACCAAATGCGGCTCGGGTCCTTGGTTGACACCTAGGTGTCTAGTTGACTACCCATACAGACTTTGGCACTACCCCTGCACTATCAATTTTACCATCTTCAAGGTCAGGATGTACGTGGGGGGCGTGGAGCACAGGCTCAACGCCGCGTGCAATTGGACCCGAGGAGAGCGCTGTGACCTGGAGGACAGGGATAGATCAGAGCTTAGCCCGCTGCTATTGTCTACAACGGAGTGGCAGGTACTGCCCTGTTCCTTTACCACCCTACCGGCTCTGTCCACTGGATTGATCCACCTCCATCAGAATATCGTGGACGTGCAATACCTGTACGGTGTAGGGTCAGTGGTTGTCTCCGTCGTAATCAAATGGGAGTATGTTCTGCTGCTCTTCCTTCTCCTGGCGGACGCGCGCGTCTGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCTGAGGCCACCTTAGAGAACCTGGTGGTCCTCAATGCGGCGTCTGTGGCCGGAGCGCATGGCCTTCTCTCCTTCCTCGTGTTCTTCTGCGCCGCCTGGTACATCAAAGGCAGGCTGGTCCCTGGGGCGGCATATGCTCTCTATGGCGTATGGCCGTTGCTCCTGCTCTTGCTGGCTTTACCACCACGAGCTTATGCCATGGACCGAGAGATGGCTGCATCGTGCGGAGGCGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCATACTATAAGGTGTTCCTCGCTAGGCTCATATGGTGGTTACAATATTTTATCACCAGGGCCGAGGCGCACTTGCAAGTGTGGGTCCCCCCTCTTAATGTTCGGGGAGGCCGCGATGCCATCATCCTCCTTACATGCGCGGTCCATCCAGAGCTAATCTTTGACATCACCAAACTCCTGCTCGCCATACTCGGTCCGCTCATGGTGCTCCAAGCTGGCATAACCAGAGTGCCGTACTTCGTGCGCGCTCAAGGGCTCATTCATGCATGCATGTTAGTGCGGAAGGTCGCTGGGGGTCATTATGTCCAAATGGCCTTCATGAAGCTGGGCGCGCTGACAGGCACGTACATTTACAACCATCTTACCCCGCTACGGGATTGGGCCCACGCGGGCCTACGAGACCTTGCGGTGGCAGTGGAGCCCGTCGTCTTCTCCGACATGGAGACCAAGATCATCACCTGGGGAGCAGACACCGCGGCGTGTGGGGACATCATCTTGGGTCTGCCCGTCTCCGCCCGAAGGGGAAAGGAGATACTCCTGGGCCCGGCCGATAGTCTTGAAGGGCGGGGGTGGCGACTCCTCGCGCCCATCACGGCCTACTCCCAACAGACGCGGGGCCTACTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAACCAGGTCGAGGGAGAGGTTCAGGTGGTTTCCACCGCAACACAATCCTTCCTGGCGACCTGCGTCAACGGCGTGTGTTGGACCGTTTACCATGGTGCTGGCTCAAAGACCTTAGCCGGCCCAAAGGGGCCAATCACCCAGATGTACACTAATGTGGACCAGGACCTCGTCGGCTGGCAGGCGCCCCCCGGGGCGCGTTCCTTGACACCATGCACCTGTGGCAGCTCAGACCTTTACTTGGTCACGAGACATGCTGACGTCATTCCGGTGCGCCGGCGGGGCGACAGTAGGGGGAGCCTGCTCTCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCTTCGGGTGGTCCACTGCTCTGCCCTTCGGGGCACGCTGTGGGCATCTTCCGGGCTGCCGTATGCACCCGGGGGGTTGCGAAGGCGGTGGACTTTGTGCCCGTAGAGTCCATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAACTCATCCCCCCCGGCCGTACCGCAGTCATTTCAAGTGGCCCACCTACACGCTCCCACTGGCAGCGGCAAGAGTACTAAAGTGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTCAATCCGTCCGTTGCCGCTACCTTAGGGTTTGGGGCGTATATGTCTAAGGCACACGGTATTGACCCCAACATCAGAACTGGGGTAAGGACCATTACCACAGGCGCCCCCGTCACATACTCTACCTATGGCAAGTTTCTTGCCGATGGTGGTTGCTCTGGGGGCGCTTATGACATCATAATATGTGATGAGTGCCATTCAACTGACTCGACTACAATCTTGGGCATCGGCACAGTCCTGGACCAAGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACACCCAAACATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTATGGCAAAGCCATCCCCATTGAAGCCATCAGGGGGGGAAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTCGCCGCAAAGCTGTCAGGCCTCGGAATCAACGCTGTGGCGTATTACCGGGGGCTCGATGTGTCCGTCATACCAACTATCGGAGACGTCGTTGTCGTGGCAACAGACGCTCTGATGACGGGCTATACGGGCGACTTTGACTCAGTGATCGACTGTAACACATGTGTCACCCAGACAGTCGACTTCAGCTTGGATCCCACCTTCACCATTGAGACGACGACCGTGCCTCAAGACGCAGTGTCGCGCTCGCAGCGGCGGGGTAGGACTGGCAGGGGTAGGAGAGGCATCTACAGGTTTGTGACTCCGGGAGAACGGCCCTCGGGCATGTTCGATTCCTCGGTCCTGTGTGAGTGCTATGACGCGGGCTGTGCTTGGTACGAGCTCACCCCCGCCGAGACCTCGGTTAGGTTGCGGGCCTACCTGAACACACCAGGGTTGCCCGTTTGCCAGGACCACCTGGAGTTCTGGGAGAGTGTCTTCACAGGCCTCACCCACATAGATGCACACTTCTTGTCCCAGACCAAGCAGGCAGGAGACAACTTCCCCTACCTGGTAGCATACCAAGCCACGGTGTGCGCCAGGGCTCAGGCCCCACCTCCATCATGGGATCAAATGTGGAAGTGTCTCATACGGCTGAAACCTACGCTGCACGGGCCAACACCCTTGCTGTACAGGCTGGGAGCCGTCCAAAATGAGGTCACCCTCACCCACCCCATAACCAAATACATCATGGCATGCATGTCGGCTGACCTGGAGGTCGTCACTAGCACCTGGGTGCTGGTGGGCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACAGGCAGTGTGGTCATTGTGGGTAGGATTATCTTGTCCGGGAGGCCGGCTATTGTTCCCGACAGGGAGCTTCTCTACCAGGAGTTCGATGAAATGGAAGAGTGCGCCACGCACCTCCCTTACATTGAGCAGGGAATGCAGCTCGCCGAGCAGTTCAAGCAGAAAGCGCTCGGGTTACTGCAAACAGCCACCAAACAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCTTGAGACATTCTGGGCGAAGCACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACTCTGCCTGGGAACCCCGCAATAGCATCATTGATGGCATTCACAGCCTCTATCACCAGCCCGCTCACCACCCAAAGTACCCTCCTGTTTAACATCTTGGGGGGGTGGGTGGCTGCCCAACTCGCCCCCCCCAGCGCCGCTTCGGCTTTCGTGGGCGCCGGCATCGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGACATTCTGGCGGGTTATGGAGCAGGAGTGGCCGGCGCGCTCGTGGCCTTTAAGGTCATGAGCGGCGAGATGCCCTCTACCGAGGACCTGGTCAATCTACTTCCTGCCATCCTCTCTCCTGGCGCCCTGGTCGTCGGGGTCGTGTGTGCAGCAATACTGCGTCGGCACGTGGGTCCGGGAGAGGGGGCTGTGCAGTGGATGAACCGGCTGATAGCGTTCGCCTCGCGGGGTAATCACGTTTCCCCCACGCACTATGTGCCTGAGAGCGACGCCGCAGCGCGTGTTACTCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGGCTCCACCAGTGGATTAATGAGGACTGCTCCACACCGTGTTCCGGCTCGTGGCTAAGGGATGTTTGGGACTGGATATGCACGGTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCAGCTACCGGGAGTCCCTTTTTTCTCGTGCCAACGCGGGTACAAGGGAGTCTGGCGGGGAGACGGCATCATGCAAACCACCTGCCCATGTGGAGCACAGATCACCGGACATGTCAAAAACGGTTCCATGAGGATCGTCGGGCCTAAGACCTGCAGCAACACGTGGCATGGAACATTCCCCATCAACGCATACACCACGGGCCCCTGCACACCCTCTCCAGCGCCAAACTATTCTAGGGCGCTGTGGCGGGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGGGATTTCCACTACGTGACGGGCATGACCACTGACAACGTAAAGTGCCCATGCCAGGTTCCGGCTCCTGAATTCTTCTCGGAGGTGGACGGAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGGCCTCTCCTACGGGAGGAGGTTACATTCCAGGTCGGGCTCAACCAATACCTGGTTGGGTCACAGCTACCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTCCATGCTCACCGACCCCTCCCACATCACAGCAGAAACGGCTAAGCGTAGGTTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCACCATGTCTCTCCGGACGCTGACCTCATCGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGGAACATCACCCGCGTGGAGTCGGAGAACAAGGTGGTAGTCCTGGACTCTTTCGACCCGCTTCGAGCGGAGGAGGATGAGAGGGAAGTATCCGTTCCGGCGGAGATCCTGCGGAAATCCAAGAAGTTCCCCGCAGCGATGCCCATCTGGGCGCGCCCGGATTACAACCCTCCACTGTTAGAGTCCTGGAAGGACCCGGACTACGTCCCTCCGGTGGTGCACGGGTGCCCGTTGCCACCTATCAAGGCCCCTCCAATACCACCTCCACGGAGAAAGAGGACGGTTGTCCTAACAGAGTCCTCCGTGTCTTCTGCCTTAGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGAATCATCGGCCGTCGACAGCGGCACGGCGACCGCCCTTCCTGACCAGGCCTCCGACGACGGTGACAAAGGATCCGACGTTGAGTCGTACTCCTCCATGCCCCCCCTTGAGGGGGAACCGGGGGACCCCGATCTCAGTGACGGGTCTTGGTCTACCGTGAGCGAGGAAGCTAGTGAGGATGTCGTCTGCTGCTCAATGTCCTACACATGGACAGGCGCCTTGATCACGCCATGCGCTGCGGAGGAAAGCAAGCTGCCCATCAACGCGTTGAGCAACTCTTTGCTGCGCCACCATAACATGGTTTATGCCACAACATCTCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTGCAAGTCCTGGACGACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTCCTATCCGTAGAGGAAGCCTGCAAGCTGACGCCCCCACATTCGGCCAAATCCAAGTTTGGCTATGGGGCAAAGGACGTCCGGAACCTATCCAGCAAGGCCGTTAACCACATCCACTCCGTGTGGAAGGACTTGCTGGAAGACACTGTGACACCAATTGACACCACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGAAAGGAGGCCGTAAGCCAGCCCGCCTTATCGTATTCCCAGATCTGGGAGTCCGTGTATGCGAGAAGATGGCCCTCTATGATGTGGTCTCCACCCTTCCTCAGGTCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTGGGCAGCGAGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAAAACCCCATGGGCTTTTCATATGACACTCGCTGTTTCGACTCAACGGTCACCGAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGAAGCCAGACAGGCCATAAAATCGCTCACAGAGCGGCTTTATATCGGGGGTCCTCTGACTAATTCAAAAGGGCAGAACTGCGGTTATCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTAACACCCTCACATGTTACTTGAAGGCCTCTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACGATGCTCGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGACGCGGCGAGCCTACGAGTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCCGGGGACCCGCCCCAACCAGAATACGACTTGGAGCTGATAACATCATGTTCCTCCAATGTGTCGGTCGCCCACGATGCATCAGGCAAAAGGGTGTACTACCTCACCCGTGATCCCACCACCCCCCTCGCACGGGCTGCGTGGGAAACAGCTAGACACACTCCAGTTAACTCCTGGCTAGGCAACATTATCATGTATGCGCCCACTTTGTGGGCAAGGATGATTCTGATGACTCACTTCTTCTCCATCCTTCTAGCACAGGAGCAACTTGAAAAAGCCCTGGACTGCCAGATCTACGGGGCCTGTTACTCCATTGAGCCACTTGACCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAGATCAATAGGGTGGCTTCATGCCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAGACATCGGGCCAGGAGCGTCCGCGCTAGGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTGGGCAGTGAAGACCAAACTCAAACTCACTCCAATCCCGGCTGCGTCCCAGCTGGACTTGTCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCTGGTTCATGCTGTGCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGGAGCTAAACACTCCAGGCCAATAGGCCATTTCCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTTTTCCTTCTTTTTCCCTTTTTCTTTCTTCCTTCTTTAATGGTGGCTCCATCTTAGCCCTAGTCACGGCTAGCTGTGAAAGGTCCGTGAGCCGCATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCAGATCATGT (SEQID NO: 6688) gi|306286|gb|M96362.1|HPCUNKCDS Hepatitis C virus mRNA,complete cdsTGCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGATATTAAGTTCCCGGGCGGTGGTCAGATCGTTGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAGCCTATCCCCAAGGCTCGCCGGCCCGAGGGCAGGGCCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAATGAGGGCTTGGGGTGGGCAGGATGGCTCCTGTCACCCCGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAAGTCGCGTAATTTGGGTAAGGTCATCGACACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCCCCTAGGGGGCGTTGCCAGGGCCCTGGCACATGGTGTCCGGGTGCTGGAGGACGGCGTGAACTATGCAACAGGGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTGGCTCTGCTGTCTTGTTTGACCACCCCAGTTTCCGCTTATGAAGTGCGTAACGCGTCCGGGATGTACCATGTCACGAACGACTGCTCCAACTCAAGCATTGTGTATGAGGCAGCGGACATGATCATGCACACTCCCGGGTGCGTGCCCTGCGTTCGGGAGGACAACTCCTCCCGTTGCTGGGTGGCACTTACTCCCACGCTCGCGGCCAGGAATGCCAGCGTCCCCACTACGACATTGCGACGCCATGTCGACTTGCTCGTTGGGGTAGCTGCTTTCTGTTCCGCTATGTACGTGGGGGACCTCTGCGGATCTGTTTTCCTTGTTTCCCAGCTGTTCACCTTTTCGCCTCGCCGGCATGAGACGGTACAGGACTGCAACTGCTCAATCTATCCCGGCCGCGTATCAGGTCACCGCATGGCCTGGGATATGATGATGAACTGGTCGCCTACAACAGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCTGTCGTGGACATGGTGACAGGGTCCCACTGGGGAATCCTGGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTAATTGCGATGCTACTCTTTGCCGGCGTTGACGGAACCACCCACGTGACAGGGGGGGCGCAAGGTCGGGCCGCTAGCTCGCTAACGTCCCTCTTTAGCCCTGGGCCGGTTCAGCACCTCCAGCTCATAAACACCAACGGCAGCTGGCATATCAACAGGACCGCCCTGAGCTGCAATGACTCCCTCAACACTGGGTTTGTTGCCGCGCTGTTCTACAAATACAGGTTCAACGCGTCCGGGTGCCCGGAGCGCTTGGCCACGTGCCGCCCCATTGATACATTCGCGCAGGGGTGGGGTCCCATCACTTACACTGAGCCTCATGATTTGGATCAGAGGCCCTATTGCTGGCACTACGCGCCTCAACCGTGTGGTATTGTGCCCACGTTGCAGGTGTGTGGCCCAGTATACTGCTTCACCCCGAGTCCTGTTGCGGTGGGGACTACCGATCGTTTCGGTGCCCCTACATACAGATGGGGGGCAAATGAGACGGACGTGCTGCTCCTTAACAACGCCGGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGACATGTGGGGGCCCCCCGTGTAACATCGGGGGGGTCGGCAACAATACCTTGACCTGCCCCACGGACTGCTTCCGAAAGCACCCCGGGGCCACTTACACCAAATGCGGTTCGGGGCCTTGGTTAACACCCAGGTGCTTAGTCGACTACCCGTACAGGCTCTGGCATTACCCCTGCACTGTCAACTTTACCATCTTTAAGGTTAGGATGTACGTGGGGGGCGCGGAGCACAGGCTCGACGCCGCATGCAACTGGACTCGGGGAGAGCGTTGTGACCTGGAGGACAGGGATAGGTCAGAGCTTAGCCCGCTGCTGCTGTCTACAACAGAGTGGCAGGTACTGCCCTGTTCCTTCACAACCCTACCGGCTCTGTCCACTGGTTTGATTCATCTCCATCAGAACATCGTGGACATACAATACCTGTACGGTATAGGGTCGGCGGTTGTCTCCTTTGCGATCAAATGGGAGTATATTGTGCTGCTCTTCCTTCTTCTGGCGGACGCGCGCGTCTGCGCTTGCTTGTGGATGATGCTGCTGGTAGCGCAAGCCGAGGCCGCCTTAGAGAACCTGGTGGTCCTCAATGCAGCGTCCGTGGCCGGAGCGCATGGCATTCTTTCCTTCATTGTGTTCTTCTGTGCTGCCTGGTACATCAAGGGCAGGCTGGTTCCCGGAGCGGCATACGCCCTCTATGGCGTATGGCCGCTGCTTCTGCTTCTGCTGGCGTTACCACCACGGGCGTACGCCATGGACCGGGAGATGGCCGCATCGTGCGGAGGCGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCACACTATAAAGTGTTCCTTGCCAGGTTCATATGGTGGCTACAATATCTCATCACCAGAACCGAAGCGCATCTGCAAGTGTGGGTCCCCCCTCTCAACGTTCGGGGGGGTCGCGATGCCATCATCCTCCTCACATGCGTGGTCCACCCAGAGCTAATCTTTGACATCACAAAATATTTGCTCGCCATATTCGGCCCGCTCATGGTGCTCCAGGCCGGCATAACTAGAGTGCCGTACTTCGTGCGCGCACAAGGGCTCATTCGTGCATGCATGTTGGCGCGGAAAGTCGTGGGGGGTCATTACGTCCAAATGGTCTTCATGAAGCTGGCCGCACTAGCAGGTACGTACGTTTATGACCATCTTACTCCACTGCGAGATTGGGCTCACACGGGCTTACGAGACCTTGCAGTGGCAGTAGAGCCCGTTGTCTTCTCTGACATGGAGACCAAAGTCATCACCTGGGGGGCAGACACCGCGGCGTGCGGGGACATCATCTTGGCCCTGCCTGCTTCCGCCCGAAGGGGGAAGGAGATACTTCTGGGACCGGCCGATAGTCTTGAAGGACAGGGGTGGCGACTCCTTGCGCCCATCACGGCCTACTCCCAACAAACGCGAGGCCTGCTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAACCAGGTTGAGGGGGAGGTTCAAGTGGTTTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAATGGCGTGTGTTGGACTGTCTTCCACGGCGCCGGCTCAAAGACCCTAGCCGGCCCAAAGGGTCCAATCACCCAAATGTACACCAATGTAGACCAGGACCTTGTTGGCTGGCCGGCACCTCCTGGGGCGCGTTCCCTGACACCATGCACTTGCGGCTCCTCGGACCTTTACCTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGGCGGGGTGACGGTAGGGGGAGCCTACTCCCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCCTCGGGTGGTCCACTGCTCTGCCCTTCGGGGCACGCTGTCGGCATACTTCCGGCTGCTGTATGCACCCGGGGGGTTGCCATGGCGGTGGAATTCATACCCGTTGAGTCTATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAATCCGTCTCCCCCGGCTGTACCGCAGACATTCCAAGTGGCCCACTTACACGCTCCCACCGGCAGCGGCAAGAGCACTAGGGTGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTAAATCCGTCCGTCGCCGCCACCTTGGGTTTTGGGGCGTATATGTCCAAGGCACATGGTATCGACCCCAACCTTAGAACTGGGGTAAGGACCATCACCACAGGTGCCCCTATCACATACTCCACCTATGGCAAGTTCCTTGCCGACGGTGGCGGCTCCGGGGGCGCCTATGACATCATAATGTGTGATGAGTGCCACTCAACTGACTCGACTACCATTTATGGCATCGGCACAGTCCTGGACCAAGCGGAGACGGCTGGAGCGCGGCTCGTGGTGCTCTCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACACCTCAATATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTACGGCAAAGCCATTCCCATCGAGGCTATCAAGGGGGGAAGGCATCTCATTTTCTGCCATTCCAAGAAGAAGTGTGACGAACTCGCCGCAAAGCTGTCAGGCCTCGGACTCAATGCCGTAGCGTATTACCGGGGTCTTGACGTGTCCGTCATACCGACCAGCGGAGACGTTGTTGTCGTGGCGACGGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACTCAGTGATCGACTGTAATACGTGTGTCACCCAGACAGTCGATTTCAGCTTGGACCCCACCTTCACCATTGAGACGACGACCGTGCCCCAAGACGCAGTGTCGCGCTCGCAGAGGCGAGGCAGGACTGGTAGGGGCAGGGCTGGCATATACAGGTTTGTGACTCCAGGAGAACGGCCCTCGGGCATGTTCGATTCTTCGGTCCTGTGTGAGTGTTATGACGCGGGTTGTGCGTGGTACGAACTCACGCCCGCTGAGACCTCGGTTAGGTTGCGGGCGTACCTAAACACACCAGGGTTGCCCGTCTGCCAGGACCATCTGGAGTTCTCGGAGGGTGTCTTCACAGGCCTCACCCACATAGATGCCCACTTCTTATCCCAGACTAAACAGGCAGGAGAGAACTTCCCCTACTTGGTAGCATACCAGGCTACAGTGTGCGCCAGGGCTCAAGCCCCACCTCCATCGTGGGATGAAATGTGGAGGTGTCTCATACGGCTGAAACCTACGCTGCACGGGCCAACACCCCTGCTGTATAGGTTAGGAGCCGTCCAAAATGAGGTCACCCTCACACACCCCATAACCAAATTCATCATGACATGTATGTCGGCTGACCTGGAGGTCGTCACCAGCACCTGGGTGCTGGTAGGCGGAGTCCTCGCAGCTCTGGCCGCGTACTGCCTGACAACAGGCAGCGTGGTCATTGTGGGCAGGATCATCCTGTCCGGGAAGCCGGCTATCATCCCCGATAGGGAAGTTCTCTACCAGGAGTTCGACGAGATGGAGGAGTGTGCCTCACACCTCCCTTACTTCGAACAGGGAATGCAGCTCGCCGAGCAATTCAAACAGAAGGCGCTCGGGTTGCTGCAAACAGCCACCAAGCAGGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCTTGAGACCTTCTGGGCGAAGCACATGTGGAACTTCATTAGTGGGATACAGTACTTGGCAGGCTTGTCCACTCTGCCTGGGAACCCCGCAATACGATCACCGATGGCATTCACAGCCTCCATCACCAGCCCGCTCACCACCCAGCATACCCTCTTGTTTAACATCTTGGGGGGATGGGTGGCTGCCCAACTCGCCCCCCCCAGCGCTGCCTCAGCTTTCGTGGGCGCCGGCATCGCTGGAGCCGCTGTTGGCACGATAGGCCTTGGGAAGGTGCTTGTGGACATTCTGGCAGGTTATGGAGCAGGGGTGGCGGGCGCACTTGTGGCCTTTAAGATCATGAGCGGCGAGATGCCTTCAGCCGAGGACATGGTCAACTTACTCCCTGCCATCCTTTCTCCCGGTGCCCTGGTCGTCGGGATTGTGTGTGCAGCAATACTGCGTCGGCATGTGGGCCCAGGGGAAGGGGCTGTGCAGTGGATGAACCGGCTGATAGCGTTCGCCTCGCGGGGTAACCACGTCTCCCCCAGGCACTATGTGCCAGAGAGCGAGCCTGCAGCGCGTGTTACCCAGATCCTTTCCAGCCTCACCATCACTCAGCTGTTGAAGAGACTCCACCAGTGGATTAATGAGGACTGCTCTACGCCATGCTCCAGCTCGTGGCTAAGGGAGATTTGGGACTGGATCTGCACGGTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGATTACCGGGAGTCCCTTTTTTCTCATGCCAACGCGGGTATAAGGGAGTCTGGCGGGGGGACGGCATCATGCACACCACCTGCCCATGCGGAGCACAGATCACCGGACACGTCAAAAACGGTTCCATGAGGATCGTTGGGCCTAAAACCTGCAGCAACACGTGGTACGGGACATTCCCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCAAACTATTCCAAGGCATTGTGGAGAGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGAGATTTTCACTACGTGACGGGCATGACCACTGACAACGTGAAGTGTCCATGCCAGGTTCCGGCCCCCGAATTCTTCACGGAGGTGGATGGAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGACCTCTCCTACGGGAGGAGGTCGTATTCCAGGTCGGGCTCCACCAGTACCTGGTCGGGTCACAGCTCCCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTCCATGCTCACTGACCCCTCCCACATTACAGCAGAGACGGCTAAGCGTAGGCTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCATCATGACTCCCCGGACGCTGACCTCATTGAGGCCAACCTCTTGTGGCGGCAAGAGATGGGCGGGAACATCACCCGCGTGGAGTCAGAGAATAAGGTGGTAATCCTGGACTCTTTCGACCCGCTCCGAGCGGAGGATGATGAGGGGGAAATATCCGTTCCGGCGGAGATCCTGCGGAAATCCAGGAAATTCCCCCCAGCGCTGCCCATATGGGCGCCGCCGGATTACAACCCTCCGCTGCTAGAGTCCTGGAAGGACCCGGACTACGTTCCTCCGGTGGTACACGGGTGCCCGTTGCCGCCCACCAAGGCCCCTCCAATACCACCTCCACGGAGGAAGAGGACGGTTGTCCTGACAGAATCCACCGTGTCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGGATCGTCGGCCATCGACAGCGGTACGGCGACCGCCCCTCCTGACCAAGCCTCCGGTGACGGCGACAGAGAGTCCGACGTTGAGTCGTTCTCCTCCATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGATCTCAGCGACGGATCTTGGTCCACCGTGAGCGAGGAGGCTAGTGAGGACGTCGTCTGCTGTTCGATGTCCTACACATGGACAGGCGCCCTGATCACGCCATGCGCTGCGGAGGAAAGCAAGTTGCCCATCAACCCGTTGAGCAATTCTTTGCTACGTCACCACAACATGGTCTATGCTACAACATCCCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTGCAAGTCCTGGACGACCACTACCGGGACGTGCTTAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTTCTATCTGTAGAAGAAGCCTGCAAACTGACGCCCCCACATTCGGCCAAATCCAAATTTGGCTACGGGGCGAAGGACGTCCGGAGCCTATCCAGCAGGGCCGTTACCCACATCCGCTCCGTGTGGAAGGACCTGCTGGAAGACACTGAAACACCAATTAGCACTACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGAAGGGAGGCCGCAAGCCAGCTCGCCTTATCGTGTTCCCAGATCTGGGAGTTCGTGTATGCGAGAAGATGGCCCTTTATGACGTGGTCTCCACCCTTCCTCAGGCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTAAGCAGCGGGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAATGCCCCATGGGCTTCTCATATGACACCCGCTGTTTTGACTCAACGGTCACTGAGAATGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGAAGCCAAACTGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACTAATTCAAAAGGGCAGAACTGCGGTTACCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTAATACCCTCACATGTTACCTGAAAGCCACTGCGGCCTGTCGAGCTGCGAAGCTCCGGGACTGCACGATGCTCGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGATGCGGCGAGCCTACGAGTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCTGGGGACCCGCCTCAACCGGAATACGACTTGGAGTTGATAACATCATGTTCCTCCAATGTGTCGGTCGCACACGATGCATCTGGTAAAAGGGTGTACTACCTCACCCGTGACCCTACCACCCCCCTTGCACGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAACATCATCATGTATGCGCCCACCTTATGGGCAAGGATGATTCTGATGACTCATTTCTTCTCCATCCTTCTAGCTCAGGAGCAACTTGAAAAAACCCTAGATTGTCAGATCTACGGGGCCTGTTACTCCATTGAACCACTTGATCTACCTCAGATCATTGAGCGACTCCATGGTCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGCGAGATCAATAGGGTGGCTTCATGCCTCAGAAAACTTGGGGTACCACCCTTGCGAGCCTGGAGACATCGGGCCAGAAGTGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTGGGCGGTGAGGACCAAGCTCAAACTCACTCCAATCCCAGCCGCGTCCCGGTTGGACTTGTCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCTGGTTCATGTTGTGCCTACTCCTACTTTCCGTGGGGGTAGGCATCTACCTGCTCCCCAACCGATGAATGGGGAGCTAAACACTCCAGGCCAATAGGCCGTTTCTC (SEQ ID NO: 6689)gi|329739|gb|L02836.1|HPCCGENOM Hepatitis C China virus complete genomeATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTCAAGTTCCCGGGCGGTGGTCAGATCGTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGACCCGAGGGCAGGACCTGGGCTCAGCCCGGGTATCCTTGGCCCCTCTATGGCAATGAGGGCTTTGGGTGGGCAGGATGGCTCCTGTCACCCCGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAGGTCGCGTAATTTGGGTAAGGTCATCGATACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCCCTTGGGGGGCGCTGCCAGGGCCCTGGCACATGGTGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAGGGAATTTGCCCGGTTGCTCTTTCTCTATCTTCCTTTTAGCCTTGCTATCCTGTTTGACCACCCCAGCTTCCGCTTACGAAGTGCGTAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGCATTGTGTATGAGGCAGCGGACCTGATCATGCATACCCCTGGGTGCGTGCCCTGCGTTCGGGAAGGCAACTCCTCCCGTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACGCCACGATCCCCACTGCGACAGTACGACGGCATGTCGATCTGCTCGTTGGGGCGGCTGCTTTCTCTTCCGCCATGTACGTGGGGGATCTCTGCGGATCTGTTTTCCTTGTCTCTCAGCTGTTCACCTTCTCGCCTCGCCGGTATGAGACAATACAGGACTGCAATTGCTCAATCTATCCCGGCCACGTAACAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCGCCTACAACAGCTCTAGTGGTGTCGCAGTTACTCCGGATCCCTCAAGCCGTCATGGACATGGTGGTGGGGGCCCACTGGGGAGTCCTGGCGGGCCTTGCCTACTATGCCATGGTGGGGAATTGGGCTAAGGTTTTGATTGTGATGCTACTCTTCGCCGGCGTTGATGGGGATACCTACGCGTCTGGGGGGGCGCAGGGCCGCTCCACCCTCGGGTTCACGTCCCTCTTTACACCTGGGGCCTCTCAGAAGATCCAGCTTATAAATACCAATGGTAGCTGGCATATCAACAGGACTGCCCTGAACTGCAATGACTCCCTCAATACTGGGTTTCTTGCCGCGCTGTTCTATACACACAGGTTCAACGCGTCCGGATGCGCAGAGCGCATGGCCAGCTGCCGCCCCATTGATACATTCGATCAGGGCTGGGGCCCCATCACTTATACTGAGCCTGATAGCTCGGACCAGAGGCCTTATTGCTGGCACTACGCGCCTCGAAAGTGCGGCATCGTACCTGCGTCGGAGGTGTGCGGTCCAGTGTATTGTTTCACCCCAAGCCCTGTCGTCGTGGGGACGACCGATCGTTTCGGTGTCCCCACATATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTCCTCAACAACACGCGGCCGCCGCAAGGCAACTGGTTTGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGACGTGCGGGGGGCCTCCGTGTAACATCGGGGGGGTCGGCAACAACACTTTGACTTGCCCCACGGATTGCTTTCGGAAGCACCCCGAGGCTACGTATACAAGGTGTGGTTCGGGGCCTTGGCTGACACCTAGGTGCTTAGTTGACTACCCATACAGGCTCTGGCACTACCCCTGCACTGTCAACTTTGCCATCTTCAAAGTTAGGATGTATGTGGGGGGCGTGGAGCACAGGCTCGATGCTGCATGCAACTGGACTCGAGGAGAGCGCTGTAACTTGGAGGACAGGGATAGATCAGAACTCAGCCCGCTGCTACTGTCTACAACAGAGTGGCAGATACTACCCTGCGCCTTCACCACCCTACCGGCTCTGTCCACTGGTTTAATCCATCTCCATCAGAACATCGTGGACGTGCAATACCTGTACGGTATAGGGTCAGCGGTTGCCTCCTTTGCAATTAAATGGGAGTATGTCTTGTTGCTTTTCCTTCTACTAGCAGACGCGCGCGTATGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCCGAGGCCGCCTTAGAGAACCTGGTGGTCCTCAATGCGGCGTCCGTGGCCGACGCGCATGGCATCCTCTCCTTCCTTGTGTTCTTTTGTGCCGCCTGGTACATTAAGGGCAGGCTGGTCCCCGGGGCAGCATACGCTTTCTACGGCGTGTGGCCGCTGCTCCTGCTCCTGCTGACATTACCACCACGAGCTTACGCCATGGACCGGGAGATGGCTGCATCGTGCGGAGGCGCGGTTTTTGTAGGTCTGGTATTCCTGACTTTGTCACCATACTACAAGGTGTTCCTCGCTAGGCTCATATGGTGGTTGCAATACTTCCTCACCATAGCCGAGGCGCACCTGCAAGTGTGGATCCCCCCTCTCAACATTCGAGGGGGCCGCGATGCCATCATCCTCCTCACGTGTGCAATCCACCCAGAGTCAATCTTTGACATCACCAAACTCCTGCTCGCCACGCTCGGTCCGCTCCTGGTGCTTCAGGCTGGCATAACTAGAGTGCCGTACTTTGTGCGCGCTCATGGGCTCATTCGCGCGTGCATGCTATTGCGGAAAGTTGCTGGGGGTCATTATGTCCAAATGGCCTTCATGAAGCTGGGCGCACTGACAGGTACGTACGTCTATAACCATCTTACTCCGCTGCAGTATTGGCCACGCGCGGGTTTACGAGAACTCGCGGTGGCAGTAGAGCCCGTCATCTTCTCTGACATGGAGACCAAGATTATCACCTGGGGGGCAGACACTGCAGCGTGTGGAGACATCATCTTGGGTTTACCCGTCTCCGCCCGAAGGGGAAAGGAGATACTCCTGGGGCCGGCCGATAGTCTTGAAGGGCAGGGGTGGCGACTCCTTGCGCCCATCACGGCCTACTCCCAACAGACGCGGGGCTTACTTGGTTGCATCATCACTAGCCTCACAGGCCGAGACAAGAACCAGGTCGAGGGGGAGGTTCAAGTGGTCTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAACGGTGTGTGTTGGACTGTCTATCATGGCGCCGGCTCAAAAACCTTAGCCGGCCCAAAGGGCCCAATCACCCAAATGTACACCAATGTAGACCAGGACCTCGTCGGCTGGCACCGGCCCCCCGGGGCGCGTTCCCTAACACCATGCACCTGCGGCAGCTCGGACCTTTACTTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGTCGAGGCGACAGTAGGGGGAGTTTACTCTCCCCCAGGCCTGTCTCCTACCTGAAGGGCTCGTCGGGGGGCCCACTGCTCTGCCCCTTCGGGCACGTTGCAGGCATCTTCCGGGCTGCTGTGTGCACCCGGGGGGTTGCGAAGGCGGTGGATTTTATACCCGTTGAGACCATGGAAACTACCATGCGGTCCCCGGTCTTCACGGACAACTCATCCCCTCCTGCCGTACCGCAGACATTCCAAGTGGCCCATCTACACGCTCCCACTGGCAGCGGCAAAAGCACCAAGGTGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTACTTGTCTTGAACCCGTCTGTTGCCGCCACTTTAGGTTTTGGGGCGTATATGTCTAAGGCACATGGTGTCGACCCCAACATTAGAACCGGGGTAAGGACCATCACCACGGGCGCCCCCATCACATACTCTACCTATGGCAAGTTCCTTGCTGATGGTGGTTGCTCTGGGGGTGCCTATGACATTATAATATGTGATGAGTGCCATTCAACTGACTCGACTACCATCTTGGGCATCGGCACGGTCCTGGACCAAGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACATCCAAACATCGAGGAGGTGGCCCTGTCCAATACTGGAGAGATCCCCTTCTATGGTAAAGCCATCCCCATCGAAGCCATCAGGGGGGGAAGGCATCTCATTTTCTGCCACTCCAAGAAGAAGTGTGACGAGCTTGCTGCAAAGCTATCATCGCTCGGGCTCAACGCTGTGGCGTACTACCGGGGGCTTGATGTGTCCGTCATACCATCTAGCGGAGACGTCGTTGTCGTGGCAACGGACGCTCTAATGACGGGCTTTACGGGCGACTTTGACTCAGTGATCGACTGTAACACATGTGTTACCCAAACAGTCGATTTCAGCTTGGACCCCACCTTCACCATCGAGACAACGACCGTGCCCCAAGACGCGGTGTCGCGCTCGCAGCGGCGAGGTAGGACTGGCAGGGGTAGGGAAGGCATCTACAGGTTTGTTACTCCAGGAGAACGGCCCTCGGGCATGTTCGACTCCTCAGTCCTGTGTGAGTGCTATGACGCGGGCTGTGCTTGGTACGAGCTCACGCCGGCTGAGACCACGGTTAGGTTGCGGGCTTACCTAAATACACCAGGGTTGCCCGTCTGCCAGGACCATCTGGAGTTCTGGGAGGGCGTCTTCACAGGTCTCACCCATATAGACGCTCACTTTCTGTCCCAGACCAAGCAAGCAGGAGACAACTTCCCCTACCTGGTAGCATACCAAGCTACAGTGTGTGCCAAGGCTCAGGCCCCACCTCCATCGTGGGATCAAATGTGGAAGTGCCTCACACGGCTAAAGCCTACGCTGCAGGGACCAACACCCCTGCTGTATAGGCTAGGAGCCGTCCAAAATGAGGTCACCCTCACACACCCCATAACTAAATACATCATGACATGCATGTCGGCTGACCTGGAGGTCGTCACCAGCACCTGGGTGCTGGTGGGCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACGGGCAGCGTGGTCATTGTGGGTAGGATTGTCTTGTCCGGAAGTCCGGCTATTGTTCCTGACAGGGAAGTTCTTTACCAAGACTTCGACGAGATGGAAGAGTGTGCCTCACACCTCCCTTACATCGAACAGGGAATGCAGCTCGCCGAGCAGTTCAAGCAGAAGGCGCTCGGGTTGCTGCAAACAGCCACCAAGCAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCTCGAGACATTTTGGGAAAAACACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACTCTGCCTGGGAACCCCGCAATGGCATCACTGATGGCATTCACAGCTTCTATCACCAGCCCGCTCACTACCCAACACACCCTCCTGTTTAACATCTTGGGTGGATGGGTGGCTGCCCAACTCGCTCCCCCCAGCGCCGCTTCGGCCTTTGTGGGCGCCGGCATTGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGACATCCTGGCGGGTTATGGGGCGGGGGTGGCTGGCGCACTCGTGGCCTTTAAGGTCATGAGTGGCGAAATGCCCTCCACTGAGGACCTGGTTAATTTACTCCCTGCCATCCTCTCTCCTGGTGCCCTAGTCGTCGGGGTCGTGTGCGCAGCAATACTGCGCCGACACGTGGGCCCGGGAGAGGGGGCTGTGCAGTGGATGAACCGGCTGATAGCGTTCGCTTCGCGGGGTAACCATGTCTCCCCCACGCACTATGTGCCTGAAAGTGACGCCGCAGCGCGTGTTACCCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGACTTCACCAGTGGATTAATGAGGACTGTTCCACACCATGCTCCGGCTCGTGGCTAAGGGATGTTTGGGATTGGATATGCACGGTGTTGACCGATTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGGTTGCCCGGAGTCCCTTTCCTCTCATGCCAACGCGGGTACAAGGGAGTCTGGCGGGGGGACGGTATTATGCAAACCACCTGTCCATGTGGAGCACAGATTACTGGACATGTCAAAAACGGTTCCATGAGAATCGTTGGGCCTAAGACTTGTAGCAACACGTGGCATGGAACATTCCCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCGAACTATTCCAGGGCGCTGTGGCGGGTGGCTCCTGAGGAGTACGTGGAGGTTACGCGGGTGGGGGATTTCCACTACGTGACGGGCATGACCACCGACAACGTGAAATGCCCATGCCAAGTCCCGGCCCCTGAATTCTTCACGGAGGTGGATGGAGTACGGCTGCACAGGTACGCTCCGGCGTGCAAACCTCTCCTACGGGAGGAGGTCGTGTTCCAGGTCGGGCTCAACCAATACCTGGTTGGATCACAGCTCCCATGCGAGCCCGAGCCGGACGTAACAGTGCTCACTTCCATGCTTACCGACCCCTCCCACATCACAGCAGAGACGGCCAAGCGTAGGCTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGCTCTTCAGCTAGCCAATTGTCTGCGCCTTCTTTGAAGGCGACATGTACTACCCATCATGACTCCCCGGACGCCGACCTCATTGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGAAACATCACCCGTGTGGAGTCAGAAAATAAGGTAGTGATCCTGGACTCTTTCGACCCGCTTCGGGCGGAGGAGGACGAGAGGGAAGTATCCGTTGCGGCGGAGATCCTGCGGAAATCCAGGAAGTTCCCCTCAGCGCTGCCCATATGGGCACGCCCAGACTACAACCCTCCACTGCTAGAGTCCTGGAAGGACCCAGATTATGTCCCTCCGGTGGTACACGGGTGCCCGTTGCCGCCTACCACGGCCCCTCCAGTACCACCTCCACGGAGAAAAAGGACGGTCGTCCTAACAGAGTCATCCGTGTCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCTGAATCGTCGGCCGTCGACAGCGGCACGGCGACTGCCCCTCCTGACGAGGCCTCCGGCGGCGGCGACAAAGGATCCGACGTTGAGTCGTACTCCTCCATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGACCTCAGCGACGGGTCCTGGTCTACCGTGAGTGAGGAGGCCAGTGAGGACGTCGTCTGCTGCTCAATGTCCTATACATGGACAGGCGCCTTGATCACGCCATGTGCTGCGGAGGAGAGCAAGCTGCCCATCAACCCGCTGAGCAACTCCTTGCTGCGTCACCACAACATGGTCTATGCTACAACATCCCGCAGTGCAAGCCTACGGCAGAAGAAGGTCGCTTTTGACAGAATGCAAGTCCTGGACGACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTCCTATCCATAGAAGAGGCCTGCAAGCTGACGCCCCCACATTCAGCCAAATCCAAATTTGGCTATGGGGCAAAAGACGTCCGGAACCTATCCAGCAAGGCCGTTAACCACATCCGCTCCGTGTGGAAGGACTTGTTGGAAGACAATGAGACACCAATCAATACCACCATCATGGCAAAAAATGAGGTTTTCTGCGTCCAACCAGAGAAAGGAGGCCGTAAGCCAGCTCGCCTTATCGTATTCCCAGACTTGGGAGTCCGTGTGTGCGAGAAGATGGCCCTTTATGACGTGGTCTCCACCCTTCCTCAGCCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTGGGCAGCGGGTCGAATTCCTGCTAAATGCCTGGAAATCAAAGGAAAACCCTATGGGCTTCTCATATGACACCCGCTGTTTTGACTCAACGGTCACTCAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGAGGCCAGACGGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACTAATTCAAAAGGGCAGAACTGCGGTTATCGCCGGTGCCGCGCAAGTGGCGTGCTGACGACCAGCTGCGGTAATACCCTTACATGTTACTTGAAGGCCTCTGCGGCCTGTCGAGCTGCGAAGCTGCAGGACTGCACGATGCTCGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACTCAAGAGGATGCGGCGAGCCTACGAGTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCTGGGGACCTGCCCCAACCAGAATACGACTTGGAGCTAATAACATCATGCTCCTCCAATGTGTCAGTCGCCCACGATGCATCTGGCAAAAGGGTGTACTACCTCACCCGTGACCCCACCATCCCCCTCGCGCGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAACATCATCATGTATGCGCCCACTCTATGGGCAAGGATGATTCTGATGACTCACTTCTTCTCCATCCTTCTAGCTCAGGAGCAACTTGAGAAAGCCCTGGATTGCCAAATCTACGGGGCCTACTACTCCATTGAGCCACTTGACCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAGATCAATAGGGTGGCGTCATGTCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAGACATCGGGCCAGAAGCGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTGGGCAGTAAAGACCAAGCTTAAACTCACTCCAATCCCGGCTGCGTCCCGGTTGGACTTGTCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGTTGGTTCATGTTGTGCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGGAGATAAACACTCCAGGCCAATAGGCCATCCC (SEQ ID NO: 6690)gi|15422182|gb|AY051292.1|Hepatitis C virus from India polyprotein mRNA,complete cdsGCCAGCCCCCTGATGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGACGCCCACAGAACGTTAAGTTCCCGGGTGGCGGCCAGATCGTTGGCGGAGTTTGCTTGTTGCCGCGCAGGGGTCCCAGAGTGGGTGTGCGCGCGACGAGGAAGACTTCCGAGCGGTCACAACCTCGCGGAAGGCGTCAGCCTATTCCCAAGGCCCGCCGACCCGAGGGCAGGTCCTGGGCGCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGCTGTGGGTGGGCAGGATGGCTCTTGTCCCCCCGCGGCTCCCGGCCTAGTCGGGGCCCCTCTGACCCCCGGCGCAGGTCACGCAATTTGGGTAAGGTCATCGATACCCTCACGTGTGGCTTCGCCGACCTCATGGGGTACATCCCGCTCGTCGGTGCTCCTCTAGGGGGCGCTGCTAGGGCTCTGGCACATGGTGTTAGGGTTCTAGAAGACGGCGTAAATTACGCAACAGGGAACCTTCCTGGTTGCTCTTTTTCTATCTTCTTGCTTGCTCTTCTCTCCTGCTTGACAGTCCCTGCTTCGGCCGTCGAAGTGCGCAACTCTTCGGGGATCTACCATGTCACCAATGATTGCCCCAATGCGTCTGTTGTGTACGAGACAGATAGCTTGATCATACATCTGCCCGGGTGTGTGCCCTGCGTACGCGAGGGCAACGCTTCGAGGTGCTGGGTCTCCCTTAGTCCTACTGTTGCCGCTAAGGATCCGGGCGTCCCCGTCAACGAGATTCGGCGTCACGTCGACCTGATTGTCGGGGCCGCTGCATTCTGTTCGGCTATGTATGTAGGGGACTTATGCGGTTCCATCTTCCTCGTTGGCCAGCTTTTCACCCTCTCCCCTAGGCGCCACTGGACAACACAAGACTGTAATTGCTCCATCTACCCAGGACATGTGACAGGCCATCGAATGGCTTGGGACATGATGATGAATTGGTCACCTACTGGCGCTTTGGTGGTAGCGCAGCTACTCCGGATCCCACAAGCCGTCTTGGATATGATAGCCGGTGCCCACTGGGGTGTCCTAGCGGGCCCGGCATACTACTCCATGGTGGGGAACTGGGCTAAGGTTTTGGTTGTGCTACTGCTCTTCGCTGGCGTCGATGCAACCACCCAAGTCACAGGTGGCACCGCGGGCCGTAATGCATATAGATTGGCTAGCCTCTTCTCCACCGGCCCCAGCCAAAATATCCAGCTCATAAACTCCAATGGCAGCTGGCACATTAACAGGACTGCCCTGAATTGCAATGACAGCCTGCACACCGGCTGGGTAGCAGCGCTGTTCTACTCCCACAAGTTCAACTCTTCGGGGCGTCCTGAGAGGATGGCTAGTTGTCGGCCTCTTACCGCCTTCGACCAAGGGTGGGGGCCCATCACTTACGGGGGGAAAGCTAGTAACGACCAGCGGCCGTATTGCTGGCACTATGCCCCACGCCCGTGCGGTATCGTGCCGGCGAAAGAGGTTTGCGGGCCTGTATACTGTTTCACACCCAGTCCCGTGGTAGTGGGGACGACGGACAAGTACGGCGTTCCTACCTACACATGGGGCGAGAATGAGACGGATGTACTGCTCCTTAACAACTCTAGGCCGCCAATAGGGAATTGGTTCGGGTGTACGTGGATGAATTCCACTGGTTTCACCAAGACGTGCGGGGCTCCTGCCTGTAACGTCGGCGGGAGCGAGACCAACACCCTGTCGTGCCCCACAGATTGCTTCCGCAGACATCCGGACGCAACATACGCTAAGTGCGGCTCTGGCCCTTGGCTTAACCCTCGATGCATGGTGGACTACCCTTACAGGCTCTGGCACTATCCCTGCACAGTCAATTACACCATATTCAAGATCAGGATGTTCGTGGGCGGGATTGAGCACAGGCTCACCGCCGCGTGCAACTGGACGCGGGGAGAGCGCTGCGACTTGGACGACAGGGATCGTGCCGAGTTGAGCCCGCTGTTGCTGTCCACCACGCAATGGCAGGTCCTCCCCTGCTCATTCACAACGCTGCCCGCCCTGTCAACTGGCCTAATACATCTCCACCAGAACATCGTGGACGTGCAGTACCTCTACGGGTTGAGCTCGGTAGTTACATCCTGGGCCATAAGGTGGGAGTATGTCGTGCTCCTTTTCTTGCTGTTAGCAGATGCCCGCATTTGTGCCTGCCTTTGGATGATGCTTCTCATATCCCAGGTAGAGGCGGCGCTGGAGAACCTGATAGTCCTCAACGCTGCTTCCCTGGCTGGGACACACGGCATCGTCCCTTTCTTCATCTTTTTTTGTGCAGCCTGGTATCTGAAAGGCAAGTGGGCCCCTGGACTCGTCTACTCCGTCTACGGAATGTGGCCGCTGCTCCTGCTTCTCCTGGCGTTGCCCCAACGGGCGTACGCCTTGGATCAGGAGTTGGCCGCGTCGTGTGGGGCCGTGGTCTTCATCAGCCTAGCGGTACTTACCCTGTCGCCGTACTACAAACAGTACATGGCCCGCGGCATCTGGTGGCTGCAGTACATGCTGACCAGAGCGGAGGCGCTCCTGCACGTCTGGGTCCCCTCGCTCAACGCCCGGGGAGGGCGTGATGGTGCCATACTGCTCATGTGTGTGCTCCACCCGCACTTGCTCTTTGACATCACCAAAATCATGCTGGCCATTCTCGGGCCCCTGTGGATCTTGCAGGCCAGTCTGCTCAGGGTGCCGTACTTCGTGCGCGCCCACGGTCTCATTAGGCTCTGCATGCTGGTGCGCAAAACAGCGGGCGGTCACTATGTGCAGATGGCTCTGTTGAAGCTGGGGGCACTTACTGGCACTTACATTTACAACCACCTTTCCCCACTCCAAGACTGGGCTCATGGCAGCTTGCGTGATCTAGCGGTGGCCACCGAGCCCGTCATCTTCTCCCGGATGGAGATCAAGACTATCACCTGGGGGGCAGACACCGCGGCCTGTGGAGACATCATCAACGGGCTGCCTGTTTCTGCTCGGAGGGGGAGAGAGGTGTTGTTGGGACCAGCCGATGCCCTGACTGACAAGGGATGGAGGCTTTTAGCCCCCATCACAGCTTACGCCCAACAGACACGAGGTCTCTTGGGCTGTATTGTCACCAGCCTCACCGGTCGGGACAAAAATCAAGTGGAGGGGGAAATCCAGATTGTGTCTACCGCAACCCAGACGTTCTTGGCCACTTGCATCAACGGAGCTTGCTGGACTGTTTATCATGGGGCCGGATCGAGGACCATCGCTTCGGCGTCGGGTCCTGTGGTCCGGATGTACACCAATGTGGACCAGGATTTGGTGGGCTGGCCAGCGCCTCAGGGAGCGCGCTCCCTGACGCCGTGCACGTGCGGTGCCTCGGATCTGTACTTGGTCACGAGGCACGCGGATGTCATCCCAGTGCGGCGTCGAGGCGATAACAGGGGAAGCTTGCTTTCTCCCCGGCCCATCTCATACCTAAAAGGATCCTCGGGAGGCCCTCTGCTCTGCCCCATGGGACATGTCGCGGGCATTTTTAGGGCCGCGGTGTGCACCCGTGGGGTTGCAAAGGCGGTCGACTTTGTGCCCGTTGAGTCCTTAGAGACCACCATGAGGTCCCCAGTGTTTACTGACAATTCCAGCCCTCCAACAGTGCCCCAGAGTTACCAGGTGGCACATCTACATGCACCCACTGGGAGTGGCAAGAGCACGAAGGTGCCGGCCGCTTACGCAGCTCAAGGGTACAAGGTACTTGTGCTGAACCCGTCTGTTGCTGCCACCTTAGGGTTCGGTGCTTATATGTCAAAGGCCCATGGGATTGACCCAAACGTCAGGACCGGCGTGAGGACCATTACCACAGGCTCCCCCATCACCTACTCCACCTACGGGAAATTTTTGGCTGATGGCGGATGCCCAGGAGGTGCGTACGACATCATAATATGTGACGAATGTCACTCAGTGGACGCCACCTCGATTCTGGGCATAGGGACCGTCTTGGACCAAGCGGAGACGGCGGGGGTTAGGCTCACTGTCCTTGCCACCGCTACACCACCTGGCTTGGTCACCGTGCCACATTCCAACATCGAGGAAGTTGCACTGTCCGCTGACGGGGAGAAACCATTTTATGGTAAGGCCATCCCCCTAAACTACATCAAGGGGGGGAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTCGCTGCAAAGCTGGTCGGTCTGGGCGTCAACGCGGTGGCCTTTTACCGTGGCCTCGACGTATCTGTCATTCCAACTACAGGAGACGTCGTTGTTGTAGCGACCGACGCCTTGATGACTGGCTTCACCGGCGATTTCGACTCTGTGATAGACTGCAACACCTGTGTCGTCCAGACAGTCGACTTCAGCCTAGACCCTATATTCTCTATTGAGACTTCCACCGTGCCCCAGGACGCCGTGTCCCGCTCCCAACGGAGGGGTAGGACCGGTCGAGGGAAGCATGGTATTTACAGATATGTGTCACCCGGGGAGCGGCCGTCTGGCATGTTCGACTCCGTGGTCCTCTGTGAGTGCTATGACGCGGGTTGTGCTTGGTACGAGCTTACACCCGCCGAGACCACAGTCAGGCTACGGGCATACCTTAACACCCCAGGATTGCCCGTGTGCCAGGACCACTTGGAGTTCTGGGAGAGTGTCTTCACCGGCCTCACCCACATAGATGCCCACTTCCTGTCCCAGACGAAACAGAGTGGGGAGAACTTCCCCTACCTAGTCGCATACCAAGCCACCGTGTGCGCTAGAGCTAGAGCTCCTCCCCCGTCATGGGACCAAATGTGGAAGTGCCTGATACGGCTCAAGCCCACCCTCACTGGGGCTACCCCATTACTATACAGACTGGGTAGTGTACAGAATGAGATCACCTTAACACACCCAATCACCCAATACATCATGGCTTGCATGTCGGCGGACCTGGAGGTCGTCACTAGCACGTGGGTGTTGGTGGGCGGCGTCCTAGCCGCTTTGGCCGCTTACTGCCTGTCCACAGGCAGCGTGGTCATAGTGGGCAGGATAATCCTAGGTGGGAAGCCGGCAGTCATACCTGACAGGGAGGTTCTCTACCGAGAGTTTGATGAGATGGAGGAGTGCGCCGCCCACGTCCCCTACCTCGAGCAGGGGATGCATTTGGCTGGACAGTTCAAGCAGAAAGCTCTCGGGTTGCTCCAGACAGCATCCAAGCAAGCGGAGACGATCACTCCCACTGTCCGCACCAACTGGCAGAAACTCGAGTCCTTCTGGGCTAAGCACATGTGGAACTTCGTTAGCGGGATACAATACCTGGCGGGCCTGTCAACGCTGCCCGGGAACCCCGCTATAGCGTCGCTGATGTCGTTTACGGCCGCGGTGACGAGTCCACTAACCACCCAGCAAACCCTCTTCTTTAACATCTTAGGGGGGTGGGTGGCGGCCCAGCTTGCTTCCCCAGCTGCCGCTACTGCTTTTGTCGGTGCTGGTATTACTGGCGCCGTTGTTGGCAGTGTGGGCCTAGGGAAGGTCCTAGTGGACATTATTGCTGGCTACGGGGCTGGTGTGGCGGGGGCCCTCGTGGCTTTCAAAATCATGAGCGGGGAGACCCCCACCACCGAGGATCTAGTCAACCTTCTGCCTGCCATCCTATCGCCAGGAGCTCTCGTTGTCGGCGTGGTGTGCGCAGCAATACTACGCCGGCACGTGGGCCCTGGCGAGGGCGCCGTGCAGTGGATGAACCGGCTGATAGCGTTTGCTTCTCGGGGTAACCACGTCTCCCCTACACACTACGTGCCGGAGAGCGACGCGTCGGCTCGTGTCACACAAATTCTCACCAGCCTCACTGTTACTCAGCTTCTGAAAAGGCTCCACGTGTGGATAAGCTCGGATTGCATCGCCCCGTGTGCTAGTTCTTGGCTTAAAGATGTCTGGGACTGGATATGCGAGGTGCTGAGCGACTTCAAGAATTGGCTGAAGGCCAAACTTGTACCACAACTGCCCGGGATCCCATTCGTATCCTGCCAACGCGGGTACCGTGGGGTCTGGCGGGGCGAGGGCATCGTGCACACTCGTTGCCCGTGTGGGGCCAATATAACTGGACATGTCAAGAACGGTTCGATGAGAATCGTCGGGCCTAAGACTTGCAGCAACACCTGGCGTGGGTCGTTCCCCATTAACGCTTACACTACAGGCCCGTGCACGCCCTCCCCGGCGCCGAACTATACGTTCGCGCTATGGAGGGTGTCTGCAGAGGAGTATGTGGAGGTAAGGCGGCTGGGGGACTTCCATTACGTCACGGGGGTGACCACTGATAAACTCAAGTGTCCATGCCAGGTCCCCTCACCCGAGTTCTTCACAGAGGTGGACGGGGTGCGCCTGCATAGGTACGCCCCCCCCTGCAAACCCCTGCTGCGAGAAGAGGTGACGTTTAGCATCGGGCTCAATGAATACTTGGTGGGGTCCCAGTTGCCCTGCGAGCCCGAGCCAGACGTAGCTGTACTGACATCAATGCTTACAGACCCCTCCCACATCACTGCAGAGACGGCAGCGCGTAGGCTGAAGCGGGGGTCTCCCCCCTCCCTGGCCAGCTCTTCCGCCAGCCAGCTGTCCGCGCCGTCACTGAAGGCAACATGCACCACTCACCACGACTCTCCAGACGCTGACCTCATAGAAGCCAACCTCCTGTGGAGACAGGAGATGGGGGGGAACATCACTAGGGTGGAGTCGGAGAACAAGATTGTCGTTCTGGATTCTTTCGACCCGCTCGTAGCGGAGGAGGATGATCGGGAGATCTCTATTCCAGCTGAGATTCTGCGGAAGTTCAAGCAGTTTCCTCCCGCTATGCCCATATGGGCACGGCCAGATTATAATCCTCCCCTTGTGGAACCGTGGAAGCGCCCGGACTATGAGCCACCCTTAGTCCACGGGTGCCCCCTACCACCTCCCAAGCCAACTCCGGTGCCGCCACCCCGGAGAAAGAGGACGGTGGTGCTGGACGAGTCTACAGTATCATCTGCTCTGGCTGAGCTTGCCACTAAGACCTTCGGCAGCTCTACAACCTCAGGCGTGACAAGTGGTGAAGCGACTGAATCGTCCCCGGCGCCCTCCTGCGGCGGTGAGCTGGACTCCGAAGCTGAATCTTACTCCTCCATGCCCCCTCTCGAGGGGGAGCCGGGGGACCCCGATCTCAGCGACGGGTCTTGGTCTACCGTGAGCAGTGATGGTGGCACGGAAGACGTTGTGTGCTGCTCGATGTCTTACTCGTGGACGGGCGCTTTAATCACGCCCTGTGCCTCAGAGGAAGCCAAGCTCCCTATCAACGCATTGAGCAACTCGCTGCTGCGCCACCACAACTTGGTGTATTCCACCACCTCTCGCAGCGCTGGCCAGAGACAGAAAAAAGTCACATTTGACAGAGTGCAAGTCCTGGACGACCATTACCGGGACGTGCTCAAGGAGGCTAAGGCCAAGGCATCCACGGTGAAGGCTAGACTGCTATCCGTTGAGGAAGCGTGTAGCCTGACGCCCCCACACTCCGCCAGATCAAAATTTGGCTATGGGGCGAAGGATGTCCGAAGCCATTCCAGTAAGGCTATACGCCACATCAACTCCGTGTGGCAGGACCTTCTGGAGGACAATACAACACCCATAGACACTACCATCATGGCAAAGAATGAGGTCTTCTGTGTGAAGCCCGAAAAGGGGGGCCGCAAGCCCGCTCGTCTTATCGTGTACCCCGACCTGGGAGTGCGCGTATGCGAGAAGAGGGCTTTGTATGACGTAGTCAAACAGCTCCCCATTGCCGTGATGGGAGCCTCCTACGGGTTCCAGTACTCACCAGCGCAGCGGGTCGACTTCCTGCTTAAAGCGTGGAAATCTAAGAAAGTCCCCATGGGGTTTTCCTATGACACCCGTTGCTTTGACTCAACAGTCACTGAGGCTGATATCCGTACGGAGGAAGACCTCTACCAATCTTGTGACCTGGCCCCTGAGGCTCGCATAGCCATAAGGTCCCTCACAGAGAGGCTTTACATCGGGGGCCCACTCACCAATTCTAAGGGACAAAACTGCGGCTATCGGCGATGCCGCGCAAGCGGCGTGCTGACCACTAGCTGCGGTAACACCATAACCTGCTTCCTCAAAGCCAGTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACCATGCTCGTGTGCGGCGACGACCTCGTCGTTATCTGTGAGAGCGCCGGTGTCCAGGAGGACGCTGCGAGCCTGAGAGCCTTCACGGAGGCTATGACCAGGTACTCCGCCCCCCCGGGAGACCCGCCTCAACCAGAATACGACTTGGAGCTTATAACATCCTGCTCCTCCAATGTGTCGGTCGCGCGCGACGGCGCTGGCAAAAGGGTCTATTATCTGACCCGTGACCCTGAGACTCCCCTCGCGCGTGCCGCTTGGGAGACAGCAAGACACACTCCAGTGAACTCCTGGCTAGGCAACATCATCATGTTTGCCCCCACTCTGTGGGTACGGATGGTCCTCATGACCCATTTTTTCTCCATACTCATAGCTCAGGAGCACCTTGGAAAGGCTCTAGATTGTGAAATCTATGGAGCCGTACACTCCGTCCAACCGTTGGACTTACCTGAAATCATCCAAAGACTCCACAGCCTCAGCGCGTTTTCGCTCCACAGTTACTCTCCAGGTGAAATCAATAGGGTGGCTGCATGCCTCAGGAAGCTTGGGGTTCCGCCCTTGCGAGCTTGGAGACACCGGGCCCGGAGCGTTCGCGCCACACTCCTATCCCAGGGGGGGAAAGCCGCTATATGCGGTAAGTACCTCTTCAACTGGGCGGTGAAAACCAAACTCAAACTCACTCCATTACCGTCCATGTCTCAGTTGGACTTGTCCAACTGGTTCACGGGCGGTTACAGCGGGGGAGACATTTATCACAGCGTGTCTCATGCCCGGCCCCGTTTGTTCCTCTGGTGCCTACTCCTACTTTCAGTAGGGGTAGGCATCTATCTCCTTCCCAACCGATAGACGGNTGGGCAACCACTCCGGGTCTTTAGGCCCTATTTAAACACTCCAGGCCTTTAGGCCCCGT (SEQ IDNO: 6691) gi|23510419|ref|NM_000043.3| Homo sapiens tumor necrosisfactor receptor superfamily, member 6 (TNFRSF6), transcript variant 1,mRNACCTACCCGCGCGCAGGCCAAGTTGCTGAATCAATGGAGCCCTCCCCAACCCGGGCGTTCCCCAGCGAGGCTTCCTTCCCATCCTCCTGACCACCGGGGCTTTTCGTGAGCTCGTCTCTGATCTCGCGCAAGAGTGACACACAGGTGTTCAAAGACGCTTCTGGGGAGTGAGGGAAGCGGTTTACGAGTGACTTGGCTGGAGCCTCAGGGGCGGGCACTGGCACGGAACACACCCTGAGGCCAGCCCTGGCTGCCCAGGCGGAGCTGCCTCTTCTCCCGCGGGTTGGTGGACCCGCTCAGTACGGAGTTGGGGAAGCTCTTTCACTTCGGAGGATTGCTCAACAACCATGCTGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATCGTCCAAAAGTGTTAATGCCCAAGTGACTGACATCAACTCCAAGGGATTGGAATTGAGGAAGACTGTTACTACAGTTGAGACTCAGAACTTGGAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGGACTGCACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGCCCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAAGGACATGGCTTAGAAGTGGAAATAAACTGCACCCGGACCCAGAATACCAAGTGCAGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAACACTGTGACCCTTGCACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAAGTGCAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTTTGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGAAAGCACAGAAAGGAAAACCAAGGTTCTCATGAATCTCCAACCTTAAATCCTGAAACAGTGGCAATAAATTTATCTGATGTTGACTTGAGTAAATATATCACCACTATTGCTGGAGTCATGACACTAAGTCAAGTTAAAGGCTTTGTTCGAAAGAATGGTGTCAATGAAGCCAAAATAGATGAGATCAAGAATGACAATGTCCAAGACACAGCAGAACAGAAAGTTCAACTGCTTCGTAATTGGCATCAACTTCATGGAAAGAAAGAAGCGTATGACACATTGATTAAAGATCTCAAAAAAGCCAATCTTTGTACTCTTGCAGAGAAAATTCAGACTATCATCCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTCAGAAATGAAATCCAAAGCTTGGTCTAGAGTGAAAAACAACAAATTCAGTTCTGAGTATATGCAATTAGTGTTTGAAAAGATTCTTAATAGCTGGCTGTAAATACTGCTTGGTTTTTTACTGGGTACATTTTATCATTTATTAGCGCTGAAGAGCCAACATATTTGTAGATTTTTAATATCTCATGATTCTGCCTCCAAGGATGTTTAAAATCTAGTTGGGAAAACAAACTTCATCAAGAGTAAATGCAGTGGCATGCTAAGTACCCAAATAGGAGTGTATGCAGAGGATGAAAGATTAAGATTATGCTCTGGCATCTAACATATGATTCTGTAGTATGAATGTAATCAGTGTATGTTAGTACAAATGTCTATCCACAGGCTAACCCCACTCTATGAATCAATAGAAGAAGCTATGACCTTTTGCTGAAATATCAGTTACTGAACAGGCAGGCCACTTTGCCTCTAAATTACCTCTGATAATTCTAGAGATTTTACCATATTTCTAAACTTTGTTTATAACTCTGAGAAGATCATATTTATGTAAAGTATATGTATTTGAGTGCAGAATTTAAATAAGGCTCTACCTCAAAGACCTTTGCACAGTTTATTGGTGTCATATTATACAATATTTCAATTGTGAATTCACATAGAAAACATTAAATTATAATGTTTGACTATTATATATGTGTATGCATTTTACTGGCTCAAAACTACCTACTTCTTTCTCAGGCATCAAAAGCATTTTGAGCAGGAGAGTATTACTAGAGCTTTGCCACCTCTCCATTTTTGCCTTGGTGCTCATCTTAATGGCCTAATGCACCCCCAAACATGGAAATATCACCAAAAAATACTTAATAGTCCACCAAAAGGCAAGACTGCCCTTAGAAATTCTAGCCTGGTTTGGAGATACTAACTGCTCTCAGAGAAAGTAGCTTTGTGACATGTCATGAACCCATGTTTGCAATCAAAGATGATAAAATAGATTCTTATTTTTCCCCCACCCCCGAAAATGTTCAATAATGTCCCATGTAAAACCTGCTACAAATGGCAGCTTATACATAGCAATGGTAAAATCATCATCTGGATTTAGGAATTGCTCTTGTCATACCCCCAAGTTTCTAAGATTTAAGATTCTCCTTACTACTATCCTACGTTTAAATATCTTTGAAAGTTTGTATTAAATGTGAATTTTAAGAAATAATATTTATATTTCTGTAAATGTAAACTGTGAAGATAGTTATAAACTGAAGCAGATACCTGGAACCACCTAAAGAACTTCCATTTATGGAGGATTTTTTTGCCCCTTGTGTTTGGAATTATAAAATATAGGTAAAAGTACGTAATTAAATAATGTTTTTGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6692)gi|35910|emb|X12387.1|HSRCYP3 Human mRNA for cytochrome P-450 (cyp3locus)GAATTCCCAAAGAGCAACACAGAGCTGAAAGGAAGACTCAGAGGAGAGAGATAAGTAAGGAAAGTAGTGATGGCTCTCATCCCAGACTTGGCCATGGAAACCTGGCTTCTCCTGGCTGTCAGCCTGGTGCTCCTCTATCTATATGGAACCCATTCACATGGACTTTTTAAGAAGCTTGGAATTCCAGGGCCCACACCTCTGCCTTTTTTGGGAAATATTTTGTCCTACCATAAGGGCTTTTGTATGTTTGACATGGAATGTCATAAAAAGTATGGAAAAGTGTGGGGCTTTTATGATGGTCAACAGCCTGTGCTGGCTATCACAGATCCTGACATGATCAAAACAGTGCTAGTGAAAGAATGTTATTCTGTCTTCACAAACCGGAGGCCTTTTGGTCCAGTGGGATTTATGAAAAGTGCCATCTCTATAGCTGAGGATGAAGAATGGAAGAGATTACGATCATTGCTGTCTCCAACCTTCACCAGTGGAAAACTCAAGGAGATGGTCCCTATCATTGCCCAGTATGGAGATGTGTTGGTGAGAAATCTGAGGCGGGAAGCAGAGACAGGCAAGCCTGTCACCTTGAAAGACGTCTTTGGGGCCTACAGCATGGATGTGATCACTAGCACATCATTTGGAGTGAACATCGACTCTCTCAACAATCCACAAGACCCCTTTGTGGAAAACACCAAGAAGCTTTTAAGATTTGATTTTTTGGATCCATTCTTTCTCTCAATAACAGTCTTTCCATTCCTCATCCCAATTCTTGAAGTATTAAATATCTGTGTGTTTCCAAGAGAAGTTACAAATTTTTTAAGAAAATCTGTAAAAAGGATGAAAGAAAGTCGCCTCGAAGATACACAAAAGCACCGAGTGGATTTCCTTCAGCTGATGATTGACTCTCAGAATTCAAAAGAAACTGAGTCCCACAAAGCTCTGTCCGATCTGGAGCTCGTGGCCCAATCAATTATCTTTATTTTTGCTGGCTATGAAACCACGAGCAGTGTTCTCTCCTTCATTATGTATGAACTGGCCACTCACCCTGATGTCCAGCAGAAACTGCAGGAGGAAATTGATGCAGTTTTACCCAATAAGGCACCACCCACCTATGATACTGTGCTACAGATGGAGTATCTTGACATGGTGGTGAATGAAACGCTCAGATTATTCCCAATTGCTATGAGACTTGAGAGGGTCTGCAAAAAAGATGTTGAGATCAATGGGATGTTCATTCCCAAAGGGTGGGTGGTGATGATTCCAAGCTATGCTCTTCACCGTGACCCAAAGTACTGGACAGAGCCTGAGAAGTTCCTCCCTGAAAGATTCAGCAAGAAGAACAAGGACAACATAGATCCTTACATATACACACCCTTTGGAAGTGGACCCAGAAACTGCATTGGCATGAGGTTTGCTCTCATGAACATGAAACTTGCTCTAATCAGAGTCCTTCAGAACTTCTCCTTCAAACCTTGTAAAGAAACACAGATCCCCCTGAAATTAAGCTTAGGAGGACTTCTTCAACCAGAAAAACCCGTTGTTCTAAAGGTTGAGTCAAGGGATGGCACCGTAAGTGGAGCCTGAATTTTCCTAAGGACTTCTGCTTTGCTCTTCAAGAAATCTGTGCCTGAGAACACCAGAGACCTCAAATTACTTTGTGAATAGAACTCTGAAATGAAGATGGGCTTCATCCAATGGACTGCATAAATAACCGGGGATTCTGTACATGCATTGAGCTCTCTCATTGTCTGTGTAGAGTGTTATACTTGGGAATATAAAGGAGGTGACCAAATCAGTGTGAGGAGGTAGATTTGGCTCCTCTGCTTCTCACGGGACTATTTCCACCACCCCCAGTTAGCACCATTAACTCCTCCTGAGCTCTGATAAGAGAATCAACATTTCTCAATAATTTCCTCCACAAATTATTAATGAAAATAAGAATTATTTTGATGGCTCTAACAATGACATTTATATCACATGTTTTCTCTGGAGTATTCTATAGTTTTATGTTAAATCAATAAAGACCACTTTACAAAAGTATTATCAGATGCTTTCCTGCACATTAAGGAGAATCTATAGAACTGAATGAGAACCAACAAGTAAATATTTTTGGTCATTGTAATCACTGTTGGCGTGGGGCCTTTGTCAGAACTAGAATTTGATTATTAACATAGGTGAAAGTTAATCCACTGTGACTTTGCCCATTGTTTAGAAAGAATATTCATAGTTTAATTATGCCTTTTTTGATCAGGCACATGGCTCACGCCTGTAATCCTAGCAGTTTGGGAGGCTGAGCCGGGTGGATCGCCTGAGGTCAGGAGTTCAAGACAAGCCTGGCCTACATGGTGAAACCCCATCTCTACTAAAAATACACAAATTAGCTAGGCATGGTGGACTCGCCTGTAATCTCACTACACAGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCGGATGTTGAAGTGAGCTGAGATTGCACCACTGCACTCCAGTCTGGGTGAGAGTGAGACTCAGTCTTAAAAAAATATGCCTTTTTGAAGCACGTACATTTTGTAACAAAGAACTGAAGCTCTTATTATATTATTAGTTTTGATTTAATGTTTTCAGCCCATCTCCTTTCATATTTCTGGGAGACAGAAAACATGTTTCCCTACACCTCTTGCTTCCATCCTCAACACCCAACTGTCTCGATGCAATGAACACTTAATAAAAAACAGTCGATTGGTCAAAAAAAAAAAAAAAAAAAAAAAAAGAATTC (SEQ ID NO: 6693)gi|339549|gb|M19154.1|HUMTGFB2A Human transforming growth factor-beta-2mRNA, complete cdsGCCCCTCCCGTCAGTTCGCCAGCTGCCAGCCCCGGGACCTTTTCATCTCTTCCCTTTTGGCCGGAGGAGCCGAGTTCAGATCCGCCACTCCGCACCCGAGACTGACACACTGAACTCCACTTCCTCCTCTTAAATTTATTTCTACTTAATAGCCACTCGTCTCTTTTTTTCCCCATCTCATTGCTCCAAGAATTTTTTTCTTCTTACTCGCCAAAGTCAGGGTTCCCTCTGCCCGTCCCGTATTAATATTTCCACTTTTGGAACTACTGGCCTTTTCTTTTTAAAGGAATTCAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATCAAAAACAACAACAACAAAAAACCAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTTTTTATTCTGACTTTTAAAAACAACTTTTTTTTCCACTTTTTTAAAAAATGCACTACTGTGTGCTGAGCGCTTTTCTGATCCTGCATCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAGTTCATGCGCAAGAGGATCGAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAGAAGACTATCCTGAGCCCGAGGAAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCTCCAGGAGAAGGCGAGCCGGAGGGCGGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACGCCAAGGAGGTTTACAAAATAGACATGCCGCCCTTCTTCCCCTCCGAAACTGTCTGCCCAGTTGTTACAACACCCTCTGGCTCAGTGGGCAGCTTGTGCTCCAGACAGTCCCAGGTGCTCTGTGGGTACCTTGATGCCATCCCGCCCACTTTCTACAGACCCTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATGGAGAAGAATGCTTCCAATTTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGCCTGAACAACGGATTGAGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGACAGCAAAGTTGTGAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGGCTTCACCATAAAGACAGGAACCTGGGATTTAAAATAAGCTTACACTGTCCCTGCTGCACTTTTGTACCATCTAATAATTACATCATCCCAAATAAAAGTGAAGAACTAGAAGCAAGATTTGCAGGTATTGATGGCACCTCCACATATACCAGTGGTGATCAGAAAACTATAAAGTCCACTAGGAAAAAAAACAGTGGGAAGACCCCACATCTCCTGCTAATGTTATTGCCCTCCTACAGACTTGAGTCACAACAGACCAACCGGCGGAAGAAGCGTGCTTTGGATGCGGCCTATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCTAGGGTGGAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTATGGAGTTCAGACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTTCTCCTTGCTGCGTGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGATTGAACAGCTTTCTAATATGATTGTAAAGTCTTGCAAATGCAGCTAAAATTCTTGGAAAAGTGGCAAGACCAAAATGACAATGATGATGATAATGATGATGACGACGACAACGATGATGCTTGTAACAAGAAAACATAAGAGAGCCTTGGTTCATCAGTGTTAAAAAATTTTTGAAAAGGCGGTACTAGTTCAGACACTTTGGAAGTTTGTGTTCTGTTTGTTAAAACTGGCATCTGACACAAAAAAAGTTGAAGGCCTTATTCTACATTTCACCTACTTTGTAAGTGAGAGAGACAAGAAGCAAATTTTTTTTAAAGAAAAAAATAAACACTGGAAGAATTTATTAGTGTTAATTATGTGAACAACGACAACAACAACAACAACAACAAACAGGAAAATCCCATTAAGTGGAGTTGCTGTACGTACCGTTCCTATCCCGCGCCTCACTTGATTTTTCTGTATTGCTATGCAATAGGCACCCTTCCCATTCTTACTCTTAGAGTTAACAGTGAGTTATTTATTGTGTGTTACTATATAATGAACGTTTCATTGCCCTTGGAAAATAAAACAGGTGTATAAAGTGGAGACCAAATACTTTGCCAGAAACTCATGGATGGCTTAAGGAACTTGAACTCAAACGAGCCAGAAAAAAAGAGGTCATATTAATGGGATGAAAACCCAAGTGAGTTATTATATGACCGAGAAAGTCTGCATTAAGATAAAGACCCTGAAAACACATGTTATGTATCAGCTGCCTAAGGAAGCTTCTTGTAAGGTCCAAAAACTAAAAAGACTGTTAATAAAAGAAACTTTCAGTCAG (SEQ ID NO: 6694)gi|186624|gb|J04111.1|HUMJUNA Human c-jun proto oncogene (JUN), completecds, clone hCJ-1CCCGGGGAGGGGACCGGGGAACAGAGGGCCGAGAGGCGTGCGGCAGGGGGGAGGGTAGGAGAAAGAAGGGCCCGACTGTAGGAGGGCAGCGGAGCATTACCTCATCCCGTGAGCCTCCGCGGGCCCAGAGAAGAATCTTCTAGGGTGGAGTCTCCATGGTGACGGGCGGGCCCGCCCCCCTGAGAGCGACGCGAGCCAATGGGAAGGCCTTGGGGTGACATCATGGGCTATTTTTAGGGGTTGACTGGTAGCAGATAAGTGTTGAGCTCGGGCTGGATAAGGGCTCAGAGTTGCACTGAGTGTGGCTGAAGCAGCGAGGCGGGAGTGGAGGTGCGCGGAGTCAGGCAGACAGACAGACACAGCCAGCCAGCCAGGTCGGCAGTATAGTCCGAACTGCAAATCTTATTTTCTTTTCACCTTCTCTCTAACTGCCCAGAGCTAGCGCCTGTGGCTCCCGGGCTGGTGGTTCGGGAGTGTCCAGAGAGCCTTGTCTCCAGCCGGCCCCGGGAGGAGAGCCCTGCTGCCCAGGCGCTGTTGACAGCGGCGGAAAGCAGCGGTACCCCACGCGCCCGCCGGGGGACGTCGGCGAGCGGCTGCAGCAGCAAAGAACTTTCCCGGCGGGGAGGACCGGAGACAAGTGGCAGAGTCCCGGAGCGAACTTTTGCAAGCCTTTCCTGCGTCTTAGGCTTCTCCACGGCGGTAAAGACCAGAAGGCGGCGGAGAGCCACGCAAGAGAAGAAGGACGTGCGCTCAGCTTCGCTCGCACCGGTTGTTGAACTTGGGCGAGCGCGAGCCGCGGCTGCCGGGCGCCCCCTCCCCCTAGCAGCGGAGGAGGGGACAAGTCGTCGGAGTCCGGGCGGCCAAGACCCGCCGCCGGCCGGCCACTGCAGGGTCCGCACTGATCCGCTCCGCGGGGAGAGCCGCTGCTCTGGGAAGTGAGTTCGCCTGCGGACTCCGAGGAACCGCTGCGCCCGAAGAGCGCTCAGTGAGTGACCGCGACTTTTCAAAGCCGGGTAGCGCGCGCGAGTCGACAAGTAAGAGTGCGGGAGGCATCTTAATTAACCCTGCGCTCCCTGGAGCGAGCTGGTGAGGAGGGCGCAGCGGGGACGACAGCCAGCGGGTGCGTGCGCTCTTAGAGAAACTTTCCCTGTCAAAGGCTCCGGGGGGCGCGGGTGTCCCCCGCTTGCCAGAGCCCTGTTGCGGCCCCGAAACTTGTGCGCGCACGCCAAACTAACCTCACGTGAAGTGACGGACTGTTCTATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCGGACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGGGAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAGCTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCGACCCCCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGGTTCGCCGAGGGCTTCGTGCGCGCCCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCAGGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGCACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGCGCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCACCTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCGAGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAGGAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTGGAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAACAGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCAGTTGCAAACATTTTGAAGAGAGACCGTCGGGGGCTGAGGGGCAACGAAGAAAAAAAATAACACAGAGAGACAGACTTGAGAACTTGACAAGTTGCGACGGAGAGAAAAAAGAAGTGTCCGAGAACTAAAGCCAAGGGTATCCAAGTTGGACTGGGTTCGGTCTGACGGCGCCCCCAGTGTGCACGAGTGGGAAGGACTTGGTCGCGCCCTCCCTTGGCGTGGAGCCAGGGAGCGGCCGCCTGCGGGCTGCCCCGCTTTGCGGACGGGCTGTCCCCGCGCGAACGGAACGTTGGACTTTCGTTAACATTGACCAAGAACTGCATGGACCTAACATTCGATCTCATTCAGTATTAAAGGGGGGAGGGGGAGGGGGTTACAAACTGCAATAGAGACTGTAGATTGCTTCTGTAGTACTCCTTAAGAACACAAAGCGGGGGGAGGGTTGGGGAGGGGCGGCAGGAGGGAGGTTTGTGAGAGCGAGGCTGAGCCTACAGATGAACTCTTTCTGGCCTGCTTTCGTTAACTGTGTATGTACATATATATATTTTTTAATTTGATTAAAGCTGATTACTGTCAATAAACAGCTTCATGCCTTTGTAAGTTATTTCTTGTTTGTTTGTTTGGGTATCCTGCCCAGTGTTGTTTGTAAATAAGAGATTTGGAGCACTCTGAGTTTACCATTTGTAATAAAGTATATAATTTTTTTATGTTTTGTTTCTGAAAATTCCAGAAAGGATATTTAAGAAAATACAATAAACTATTGGAAAGTACTCCCCTAACCTCTTTTCTGCATCATCTGTAGATCCTAGTCTATCTAGGTGGAGTTGAAAGAGTTAAGAATGCTCGATAAAATCACTCTCAGTGCTTCTTACTATTAAGCAGTAAAAACTGTTCTCTATTAGACTTAGAAATAAATGTACCTGATGTACCTGATGCTATGTCAGGCTTCATACTCCACGCTCCCCCAGCGTATCTATATGGAATTGCTTACCAAAGGCTAGTGCGATGTTTCAGGAGGCTGGAGGAAGGGGGGTTGCAGTGGAGAGGGACAGCCCACTGAGAAGTCAAACATTTCAAAGTTTGGATTGCATCAAGTGGCATGTGCTGTGACCATTTATAATGTTAGAAATTTTACAATAGGTGCTTATTCTCAAAGCAGGAATTGGTGGCAGATTTTACAAAAGATGTATCCTTCCAATTTGGAATCTTCTCTTTGACAATTCCTAGATAAAAAGATGGCCTTTGTCTTATGAATATTTATAACAGCATTCTGTCACAATAAATGTATTCAAATACCAATAACAGATCTTGAATTGCTTCCCTTTACTACTTTTTTGTTCCCAAGTTATATACTGAAGTTTTTATTTTTAGTTGCTGAGGTT (SEQ ID NO: 6695)gi|179982|gb|M57729.1|HUMCCC5 Human complement component C5 mRNA,complete cdsCTACCTCCAACCATGGGCCTTTTGGGAATACTTTGTTTTTTAATCTTCCTGGGGAAAACCTGGGGACAGGAGCAAACATATGTCATTTCAGCACCAAAAATATTCCGTGTTGGAGCATCTGAAAATATTGTGATTCAAGTTTATGGATACACTGAAGCATTTGATGCAACAATCTCTATTAAAAGTTATCCTGATAAAAAATTTAGTTACTCCTCAGGCCATGTTCATTTATCCTCAGAGAATAAATTCCAAAACTCTGCAATCTTAACAATACAACCAAAACAATTGCCTGGAGGACAAAACCCAGTTTCTTATGTGTATTTGGAAGTTGTATCAAAGCATTTTTCAAAATCAAAAAGAATGCCAATAACCTATGACAATGGATTTCTCTTCATTCATACAGACAAACCTGTTTATACTCCAGACCAGTCAGTAAAAGTTAGAGTTTATTCGTTGAATGACGACTTGAAGCCAGCCAAAAGAGAAACTGTCTTAACCTTCATAGATCCTGAAGGATCAGAAGTTGACATGGTAGAAGAAATTGATCATATTGGAATTATCTCTTTTCCTGACTTCAAGATTCCGTCTAATCCTAGATATGGTATGTGGACGATCAAGGCTAAATATAAAGAGGACTTTTCAACAACTGGAACCGCATATTTTGAAGTTAAAGAATATGTCTTGCCACATTTTTCTGTCTCAATCGAGCCAGAATATAATTTCATTGGTTACAAGAACTTTAAGAATTTTGAAATTACTATAAAAGCAAGATATTTTTATAATAAAGTAGTCACTGAGGCTGACGTTTATATCACATTTGGAATAAGAGAAGACTTAAAAGATGATCAAAAAGAAATGATGCAAACAGCAATGCAAAACACAATGTTGATAAATGGAATTGCTCAAGTCACATTTGATTCTGAAACAGCAGTCAAAGAACTGTCATACTACAGTTTAGAAGATTTAAACAACAAGTACCTTTATATTGCTGTAACAGTCATAGAGTCTACAGGTGGATTTTCTGAAGAGGCAGAAATACCTGGCATCAAATATGTCCTCTCTCCCTACAAACTGAATTTGGTTGCTACTCCTCTTTTCCTGAAGCCTGGGATTCCATATCCCATCAAGGTGCAGGTTAAAGATTCGCTTGACCAGTTGGTAGGAGGAGTCCCAGTAATACTGAATGCACAAACAATTGATGTAAACCAAGAGACATCTGACTTGGATCCAAGCAAAAGTGTAACACGTGTTGATGATGGAGTAGCTTCCTTTGTGCTTAATCTCCCATCTGGAGTGACGGTGCTGGAGTTTAATGTCAAAACTGATGCTCCAGATCTTCCAGAAGAAAATCAGGCCAGGGAAGGTTACCGAGCAATAGCATACTCATCTCTCAGCCAAAGTTACCTTTATATTGATTGGACTGATAACCATAAGGCTTTGCTAGTGGGAGAACATCTGAATATTATTGTTACCCCCAAAAGCCCATATATTGACAAAATAACTCACTATAATTACTTGATTTTATCCAAGGGCAAAATTATCCATTTTGGCACGAGGGAGAAATTTTCAGATGCATCTTATCAAAGTATAAACATTCCAGTAACACAGAACATGGTTCCTTCATCCCGACTTCTGGTCTATTATATCGTCACAGGAGAACAGACAGCAGAATTAGTGTCTGATTCAGTCTGGTTAAATATTGAAGAAAAATGTGGCAACCAGCTCCAGGTTCATCTGTCTCCTGATGCAGATGCATATTCTCCAGGCCAAACTGTGTCTCTTAATATGGCAACTGGAATGGATTCCTGGGTGGCATTAGCAGCAGTGGACAGTGCTGTGTATGGAGTCCAAAGAGGAGCCAAAAAGCCCTTGGAAAGAGTATTTCAATTCTTAGAGAAGAGTGATCTGGGCTGTGGGGCAGGTGGTGGCCTCAACAATGCCAATGTGTTCCACCTAGCTGGACTTACCTTCCTCACTAATGCAAATGCAGATGACTCCCAAGAAAATGATGAACCTTGTAAAGAAATTCTCAGGCCAAGAAGAACGCTGCAAAAGAAGATAGAAGAAATAGCTGCTAAATATAAACATTCAGTAGTGAAGAAATGTTGTTACGATGGAGCCTGCGTTAATAATGATGAAACCTGTGAGCAGCGAGCTGCACGGATTAGTTTAGGGCCAAGATGCATCAAAGCTTTCACTGAATGTTGTGTCGTCGCAAGCCAGCTCCGTGCTAATATCTCTCATAAAGACATGCAATTGGGAAGGCTACACATGAAGACCCTGTTACCAGTAAGCAAGCCAGAAATTCGGAGTTATTTTCCAGAAAGCTGGTTGTGGGAAGTTCATCTTGTTCCCAGAAGAAAACAGTTGCAGTTTGCCCTACCTGATTCTCTAACCACCTGGGAAATTCAAGGCATTGGCATTTCAAACACTGGTATATGTGTTGCTGATACTGTCAAGGCAAAGGTGTTCAAAGATGTCTTCCTGGAAATGAATATACCATATTCTGTTGTACGAGGAGAACAGATCCAATTGAAAGGAACTGTTTACAACTATAGGACTTCTGGGATGCAGTTCTGTGTTAAAATGTCTGCTGTGGAGGGAATCTGCACTTCGGAAAGCCCAGTCATTGATCATCAGGGCACAAAGTCCTCCAAATGTGTGCGCCAGAAAGTAGAGGGCTCCTCCAGTCACTTGGTGACATTCACTGTGCTTCCTCTGGAAATTGGCCTTCACAACATCAATTTTTCACTGGAGACTTGGTTTGGAAAAGAAATCTTAGTAAAAACATTACGAGTGGTGCCAGAAGGTGTCAAAAGGGAAAGCTATTCTGGTGTTACTTTGGATCCTAGGGGTATTTATGGTACCATTAGCAGACGAAAGGAGTTCCCATACAGGATACCCTTAGATTTGGTCCCCAAAACAGAAATCAAAAGGATTTTGAGTGTAAAAGGACTGCTTGTAGGTGAGATCTTGTCTGCAGTTCTAAGTCAGGAAGGCATCAATATCCTAACCCACCTCCCCAAAGGGAGTGCAGAGGCGGAGCTGATGAGCGTTGTCCCAGTATTCTATGTTTTTCACTACCTGGAAACAGGAAATCATTGGAACATTTTTCATTCTGACCCATTAATTGAAAAGCAGAAACTGAAGAAAAAATTAAAAGAAGGGATGTTGAGCATTATGTCCTACAGAAATGCTGACTACTCTTACAGTGTGTGGAAGGGTGGAAGTGCTAGCACTTGGTTAACAGCTTTTGCTTTAAGAGTACTTGGACAAGTAAATAAATACGTAGAGCAGAACCAAAATTCAATTTGTAATTCTTTATTGTGGCTAGTTGAGAATTATCAATTAGATAATGGATCTTTCAAGGAAAATTCACAGTATCAACCAATAAAATTACAGGGTACCTTGCCTGTTGAAGCCCGAGAGAACAGCTTATATCTTACAGCCTTTACTGTGATTGGAATTAGAAAGGCTTTCGATATATGCCCCCTGGTGAAAATCGACACAGCTCTAATTAAAGCTGACAACTTTCTGCTTGAAAATACACTGCCAGCCCAGAGCACCTTTACATTGGCCATTTCTGCGTATGCTCTTTCCCTGGGAGATAAAACTCACCCACAGTTTCGTTCAATTGTTTCAGCTTTGAAGAGAGAAGCTTTGGTTAAAGGTAATCCACCCATTTATCGTTTTTGGAAAGACAATCTTCAGCATAAAGACAGCTCTGTACCTAACACTGGTACGGCACGTATGGTAGAAACAACTGCCTATGCTTTACTCACCAGTCTGAACTTGAAAGATATAAATTATGTTAACCCAGTCATCAAATGGCTATCAGAAGAGCAGAGGTATGGAGGTGGCTTTTATTCAACCCAGGACACCATCAATGCCATTGAGGGCCTGACGGAATATTCACTCCTGGTTAAACAACTCCGCTTGAGTATGGACATCGATGTTTCTTACAAGCATAAAGGTGCCTTACATAATTATAAAATGACAGACAAGAATTTCCTTGGGAGGCCAGTAGAGGTGCTTCTCAATGATGACCTCATTGTCAGTACAGGATTTGGCAGTGGCTTGGCTACAGTACATGTAACAACTGTAGTTCACAAAACCAGTACCTCTGAGGAAGTTTGCAGCTTTTATTTGAAAATCGATACTCAGGATATTGAAGCATCCCACTACAGAGGCTACGGAAACTCTGATTACAAACGCATAGTAGCATGTGCCAGCTACAAGCCCAGCAGGGAAGAATCATCATCTGGATCCTCTCATGCGGTGATGGACATCTCCTTGCCTACTGGAATCAGTGCAAATGAAGAAGACTTAAAAGCCCTTGTGGAAGGGGTGGATCAACTATTCACTGATTACCAAATCAAAGATGGACATGTTATTCTGCAACTGAATTCGATTCCCTCCAGTGATTTCCTTTGTGTACGATTCCGGATATTTGAACTCTTTGAAGTTGGGTTTCTCAGTCCTGCCACTTTCACAGTTTACGAATACCACAGACCAGATAAACAGTGTACCATGTTTTATAGCACTTCCAATATCAAAATTCAGAAAGTCTGTGAAGGAGCCGCGTGCAAGTGTGTAGAAGCTGATTGTGGGCAAATGCAGGAAGAATTGGATCTGACAATCTCTGCAGAGACAAGAAAACAAACAGCATGTAAACCAGAGATTGCATATGCTTATAAAGTTAGCATCACATCCATCACTGTAGAAAATGTTTTTGTCAAGTACAAGGCAACCCTTCTGGATATCTACAAAACTGGGGAAGCTGTTGCTGAGAAAGACTCTGAGATTACCTTCATTAAAAAGGTAACCTGTACTAACGCTGAGCTGGTAAAAGGAAGACAGTACTTAATTATGGGTAAAGAAGCCCTCCAGATAAAATACAATTTCAGTTTCAGGTACATCTACCCTTTAGATTCCTTGACCTGGATTGAATACTGGCCTAGAGACACAACATGTTCATCGTGTCAAGCATTTTTAGCTAATTTAGATGAATTTGCCGAAGATATCTTTTTAAATGGATGCTAAAATTCCTGAAGTTCAGCTGCATACAGTTTGCACTTATGGACTCCTGTTGTTGAAGTTCGTTTTTTTGTTTTCTTCTTTTTTTAAACATTCATAGCTGGTCTTATTTGTAAAGCTCACTTTACTTAGAATTAGTGGCACTTGCTTTTATTAGAGAATGATTTCAAATGCTGTAACTTTCTGAAATAACATGGCCTTGGAGGGCATGAAGACAGATACTCCTCCAAGGTTATTGGACACCGGAAACAATAAATTGGAACACCTCCTCAAACCTACCACTCAGGAATGTTTGCTGGGGCCGAAAGAACAGTCCATTGAAAGGGAGTATTACAAAAACATGGCCTTTGCTTGAAAGAAAATACCAAGGAACAGGAAACTGATCATTAAAGCCTGAGTTTGCTTTC (SEQ ID NO: 6696)gi|189944|gb|L05144.1|HUMPHOCAR Homo sapiens (clone lamda-hPEC-3)phosphoenolpyruvate carboxykinase (PCK1) mRNA, complete cdsTGGGAACACAAACTTGCTGGCGGGAAGAGCCCGGAAAGAAACCTGTGGATCTCCCTTCGAGATCATCCAAAGAGAAGAAAGGTGACCTCACATTCGTGCCCCTTAGCAGCACTCTGCAGAAATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTGCCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAATGGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAGAAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGGGGCCGCTGGGCTCACCTCTGTCGAAGATCGGCATCGAGCTGACGGATTCGCCCTACGTGGTGGCCAGCATGCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCACTGGGCGATGGGGAGTTTGTCAAATGCCTCCATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCTGCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCAGAGGAGGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGATGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAATGGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGGCGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAACTCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTCCCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTGGCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATCTTCCATGTCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACCAACGTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGAAAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAATTCTGGGACAAGGAGGTGGAAGACATCGAGAAGTATCTGGTGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGTAATCAGGGCCTGAGAATAAGCCAGATGTAATCAGGGCCTGAGTGCTTTACCTTTAAAATCATTAAATTAAAATCCATAAGGTGCAGTAGGAGCAAGAGAGGGCAAGTGTTCCCAAATTGACGCCACCTAATAATCATCACCACACCGGGAGCAGATCTGAAGGCACACTTTGATTTTTTTAAGGATAAGAACCACAGAACACTGGGTAGTAGCTAATGAAATTGAGAAGGGAAATCTTAGCATGCCTCCAAAAATTCACATCCAATGCATACTTTGTTCAAATTTAAGGTTACTCAGGCATTGATCTTTTCAGTGTTTTTTCACTTAGCTATGTGGATTAGCTAGAATGCACACCAAAAAGATACTTGAGCTGTATATATATATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCATGTATGTGCACATGTGTCTGTGTGATATTTGGTATGTGTATTTGTATGTACTGTTATTCAAAATATATTTAATACCTTTGGAAAATCTTGGGCAAGATGACCTACTAGTTTTCCTTGAAAAAAAGTTGCTTTGTTATTAATATTGTGCTTAAATTATTTTTATACACCATTGTTCCTTACCTTTACATAATTGCAATATTTCCCCCTTACTACTTCTTGGAAAAAAATTAGAAAATGAAGTTTATAGAAAAG (SEQID NO: 6697) gi|6679892|ref|NM_008061.1| Mus musculusglucose-6-phosphatase, catalytic (G6pc), mRNAAGCAGAGGGATCGGGGCCAACCGGGCTTGGACTCACTGCACGGGCTCTGCTGGCAGCTTCCTGAGGTACCAAGGGAGGAAGGATGGAGGAAGGAATGAACATTCTCCATGACTTTGGGATCCAGTCGACTCGCTATCTCCAAGTGAATTACCAAGACTCCCAGGACTGGTTCATCCTTGTGTCTGTGATTGCTGACCTGAGGAACGCCTTCTATGTCCTCTTTCCCATCTGGTTCCATCTTAAAGAGACTGTGGGCATCAATCTCCTCTGGGTGGCAGTGGTCGGAGACTGGTTCAACCTCGTCTTCAAGTGGATTCTGTTTGGACAACGCCCGTATTGGTGGGTCCTGGACACCGACTACTACAGCAACAGCTCCGTGCCTATAATAAAGCAGTTCCCTGTCACCTGTGAGACCGGACCAGGAAGTCCCTCTGGCCATGCCATGGGCGCAGCAGGTGTATACTATGTTATGGTCACTTCTACTCTTGCTATCTTTCGAGGAAAGAAAAAGCCAACGTATGGATTCCGGTGTTTGAACGTCATCTTGTGGTTGGGATTCTGGGCTGTGCAGCTGAACGTCTGTCTGTCCCGGATCTACCTTGCTGCTCACTTTCCCCACCAGGTCGTGGCTGGAGTCTTGTCAGGCATTGCTGTGGCTGAAACTTTCAGCCACATCCGGGGCATCTACAATGCCAGCCTCCGGAAGTATTGTCTCATCACCATCTTCTTGTTTGGTTTCGCGCTTGGATTCTACCTGCTACTAAAAGGGCTAGGGGTGGACCTCCTGTGGACTTTGGAGAAAGCCAAGAGATGGTGTGAGCGGCCAGAATGGGTCCACCTTGACACTACACCCTTTGCCAGCCTCTTCAAAAACCTGGGAACCCTCTTGGGGTTGGGGCTGGCCCTCAACTCCAGCATGTACCGGAAGAGCTGCAAGGGAGAACTCAGCAAGTCGTTCCCATTCCGCTTCGCCTGCATTGTGGCTTCCTTGGTCCTCCTGCATCTCTTTGACTCTCTGAAGCCCCCATCCCAGGTTGAGTTGATCTTCTACATCTTGTCTTTCTGCAAGAGCGCAACAGTTCCCTTTGCATCTGTCAGTCTTATCCCATACTGCCTAGCCCGGATCCTGGGACAGACACACAAGAAGTCTTTGTAAGGCATGCAGAGTCTTTGGTATTTAAAGTCAACCGCCATGCAAAGGACTAGGAACAACTAAAGCCTCTGAAACCCATTGTGAGGCCAGAGGTGTTGACATCGGCCCTGGTAGCCCTGTCTTTCTTTGCTATCTTAACCAAAAGGTGAATTTTTACAAAGCTTACAGGGCTGTTTGAGGAAAGTGTGAATGCTGGAAACTGAGTCATTCTGGATGGTTCCCTGAAGATTCGCTTACCAGCCTCCTGTCAGATACAGAAGAGCAAGCCCAGGCTAGAGATCCCAACTGAGAATGCTCTTGCGGTGCAGAATCTTCCGGCTGGGAAAAGGAAAAGAGCACCATGCATTTGCCAGGAAGAGAAAGAAGGATCGGGAGGAGGGAGAGTGTTTTATGTATCGAGCAAACCAGATGCAATCTATGTCTAACCGGCTTCAGTTGTGTCTGCGTCTTTAGATACGACACACTCAATAATAATAATAGACCAACTAGTGTAATGAGTAGCCAGTTAAAGGCGATTAATTCTGCTTCCAGATAGTCTCCACTGTACATAAAAGTCACACTGTGTGCTTGCATTCCTGTATGGTAGTGGTGACTGTCTCTCACACCACCTTCTCTATCACGTCACAGTTTTCTCCTCCTCAGCCTATGTCTGCATTCCCCAGAATTCTCCACTTGTTCCCTGGCCCTGCTGCTGGACCCTGCTGTGTCTGGTAGGCAACTGTTTGTTGGTGCTTTTGTAGGGTTAAGTTAAACTCTGAGATCTTGGGCAAAATGGCAAGGAGACCCAGGATTCTTCTCTCCAAAGGTCACTCCGATGTTATTTTTGATTCCTGGGGCAGAAATATGACTCCTTTCCCTAGCCCAAGCCAGCCAAGAGCTCTCATTCTTAGAAGAAAAGGCAGCCCCTTGGTGCCTGTCCTCCTGCCTCGGCTGATTTGCAGAGTACTTCTTCAAAAAGAAAAAAATGGTAAAGCTATTTATTAAAAATTCTTTGTTTTTTGCTACAAATGATGCATATATTTTCACCCACACCAAGCACTTTGTTTCTAATATCTTTGATAAGAAAACTACATGTGCAGTATTTTATTAAAGCAACATTTTATTTA (SEQ ID NO: 6698) gi|7110682|ref|NM_011044.1| Musmusculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNAACAGTTGGCCTTCCCTCTGGGAACACACCCTCGGTCAACAGGGGAAATCCGGCAAGGCGCTCAGCGATCTCTGATCCAGACCTTCCAAAAGGAAGAAAGGTGGCACCAGAGTTCCTGCCTCTCTCCACACCATTGCAATTATGCCTCCTCAGCTGCATAACGGTCTGGACTTCTCTGCCAAGGTTATCCAGGGCAGCCTCGACAGCCTGCCCCAGGCAGTGAGGAAGTTCGTGGAAGGCAATGCTCAGCTGTGCCAGCCGGAGTATATCCACATCTGCGATGGCTCCGAGGAGGAGTACGGGCAGTTGCTGGCCCACATGCAGGAGGAGGGTGTCATCCGCAAGCTGAAGAAATATGACAACTGTTGGCTGGCTCTCACTGACCCTCGAGATGTGGCCAGGATCGAAAGCAAGACAGTCATCATCACCCAAGAGCAGAGAGACACAGTGCCCATCCCCAAAACTGGCCTCAGCCAGCTGGGCCGCTGGATGTCGGAAGAGGACTTTGAGAAAGCATTCAACGCCAGGTTCCCAGGGTGCATGAAAGGCCGCACCATGTATGTCATCCCATTCAGCATGGGGCCACTGGGCTCGCCGCTGGCCAAGATTGGTATTGAACTGACAGACTCGCCCTATGTGGTGGCCAGCATGCGGATCATGACTCGGATGGGCATATCTGTGCTGGAGGCCCTGGGAGATGGGGAGTTCATCAAGTGCCTGCACTCTGTGGGGTGCCCTCTCCCCTTAAAAAAGCCTTTGGTCAACAACTGGGCCTGCAACCCTGAGCTGACCCTGATCGCCCACCTCCCGGACCGCAGAGAGATCATCTCCTTTGGAAGCGGATATGGTGGGAACTCACTACTCGGGAAGAAATGCTTTGCGTTGCGGATCGCCAGCCGTCTGGCTAAGGAGGAAGGGTGGCTGGCGGAGCATATGCTGATCCTGGGCATAACTAACCCCGAAGGCAAGAAGAAATACCTGGCCGCAGCCTTCCCTAGTGCCTGTGGGAAGACTAACTTGGCCATGATGAACCCCAGCCTGCCCGGGTGGAAGGTCGAATGTGTGGGCGATGACATTGCCTGGATGAAGTTTGATGCCCAAGGCAACTTAAGGGCTATCAACCCAGAAAACGGGTTTTTTGGAGTTGCTCCTGGCACCTCAGTGAAGACAAATCCAAATGCCATTAAAACCATCCAGAAAAACACCATCTTCACCAACGTGGCCGAGACTAGCGATGGGGGTGTTTACTGGGAAGGCATCGATGAGCCGCTGGCCCCGGGAGTCACCATCACCTCCTGGAAGAACAAGGAGTGGAGACCGCAGGACGCGGAACCATGTGCCCATCCCAACTCGAGATTCTGCACCCCTGCCAGCCAGTGCCCCATTATTGACCCTGCCTGGGAATCTCCAGAAGGAGTACCCATTGAGGGTATCATCTTTGGTGGCCGTAGACCTGAAGGTGTCCCCCTTGTCTATGAAGCCCTCAGCTGGCAGCATGGGGTGTTTGTAGGAGCAGCCATGAGATCTGAGGCCACAGCTGCTGCAGAACACAAGGGCAAGATCATCATGCACGACCCCTTTGCCATGCGACCCTTCTTCGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTGAGCATGGCCCACCGCCCAGCAGCCAAGTTGCCCAAGATCTTCCATGTCAACTGGTTCCGGAAGGACAAAGATGGCAAGTTCCTCTGGCCAGGCTTTGGCGAGAACTCCCGGGTGCTGGAGTGGATGTTCGGGCGGATTGAAGGGGAAGACAGCGCCAAGCTCACGCCCATCGGCTACATCCCTAAGGAAAACGCCTTGAACCTGAAAGGCCTGGGGGGCGTCAACGTGGAGGAGCTGTTTGGGATCTCTAAGGAGTTCTGGGAGAAGGAGGTGGAGGAGATCGACAGGTATCTGGAGGACCAGGTCAACACCGACCTCCCTTACGAAATTGAGAGGGAGCTCCGAGCCCTGAAACAGAGAATCAGCCAGATGTAAATCCCAATGGGGGCGTCTCGAGAGTCACCCCTTCCCACTCACAGCATCGCTGAGATCTAGGAGAAAGCCAGCCTGCTCCAGCTTTGAGATAGCGGCACAATCGTGAGTAGATCAGAAAAGCACCTTTTAATAGTCAGTTGAGTAGCACAGAGAACAGGCTAGGGGCAAATAAGATTGGGAGGGGAAATCACCGCATAGTCTCTGAAGTTTGCATTTGACACCAATGGGGGTTTTGGTTCCACTTCAAGGTCACTCAGGAATCCAGTTCTTCACGTTAGCTGTAGCAGTTAGCTAAAATGCACAGAAAACATACTTGAGCTGTATATATGTGTGTGAACGTGTCTCTGTGTGAGCATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACATGCCTGTCTGTCCCATTGTCCACAGTATATTTAAAACCTTTGGGGAAAAATCTTGGGCAAATTTGTAGCTGTAACTAGAGAGTCATGTTGCTTTGTTGCTAGTATGTATGTTTAAATTATTTTTATACACCGCCCTTACCTTTCTTTACATAATTGAAATTGGTATCCGGACCACTTCTTGGGAAAAAAATTACAAAATAAA (SEQ ID NO: 6699)

Example 6 siRNAs Decrease mRNA Levels In Vivo

Male CMV-Luc mice (8-10 weeks old) from Xenogen (Cranbury, N.J.) wereadministered cholesterol conjugated siRNA (see Table 16).

TABLE 16 Solutions adminstered to mice Group n Injection Mix 1 7 Buffer(PBS [pH 7.4]) 2 8 Cholesterol conjugated siRNA (ALN-3001)

TABLE 17 Test iRNA agents targeting Luciferase siRNA Sequence ALN-10705′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6700) 3′-CG CUU GAC ACACAC UCU CCA GGA-5′ (SEQ ID NO: 6701) ALN-1000 5′-GAA CUG UGU GUG AGA GGUCCU-GS-3′ (SEQ ID NO: 6702) 3′-CG CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ IDNO: 6703) ALN-3000 5′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6704)3′-Cs¹Gs¹ CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6705) ALN-30015′-GAA CUG UGU GUG AGA GGU CCU-chol.²-3′ (SEQ ID NO: 6706) 3′-Cs¹Gs¹ CUUGAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6707) ¹2′O-Me group is attachedto the nucleotide and the nucleotides have phosphorothioate linkages(indicated by “s”) ²cholesterol is conjugated to the antisense strandvia the linker: U-pyrroline carrier-C(O)—(CH₂)₅—NHC(O)-cholesterol (viacholesterol C-3 hydroxyl).

Animals were injected (tail vein) with a volume of 200-250 μl testsolution containing buffer or an siRNA solution. Group 1 received bufferand group 2 received cholesterol conjugated siRNA (ALN-3001) at a doseof 50 mg/kg body weight. Twenty-two hours after injection, animals weresacrificed and livers collected. Organs were snap frozen on dry ice,then pulverized in a mortar and pestle.

For Luciferase mRNA analysis (by the QuantiGene Assay (Genospectra,Inc.; Fremont, Calif.)), approximately 10 mg of tissue powder wasresuspended in tissue lysis buffer, and processed according to themanufacturer's protocol. Samples of the lysate were hybridized withprobes specific for Luciferase or GAPDH (designed using ProbeDesignersoftware (Genospectra, Inc., Fremont, Calif.) in triplicate, andprocessed for luminometric analysis. Values for Luciferase werenormalized to GAPDH. Mean values were plotted with error barscorresponding to the standard deviation of the Luciferase measurements.

Results indicated that the level of luciferase RNA in animals injectedwith cholesterol conjugated siRNA was reduced by about 70% as comparedto animals injected with buffer (see FIGS. 6A and 6 b).

In Vitro Activity

HeLa cells expressing luciferase were transfected with each of thesiRNAs listed in Table 17. ALN-1000 siRNAs were most effective atdecreasing luciferase mRNA levels (˜0.6 nM siRNA decreased mRNA levelsto about ˜65% the original expression level, and 1.0 nM siRNA decreasedlevels to about ˜20% the original expression level); ALN-3001 siRNAswere least effective (˜0.6 nM siRNA had a negligible mRNA levels, and1.0 nM siRNA decreased levels to about ˜40% the original expressionlevel).

Pharmacokinetics/Biodistribution

Pharmacokinetic analyses were performed in mice and rats. Test siRNAmolecules were radioactively labeled with ³³P on the antisense strand bysplint ligation. Labeled siRNAs (50 mg/kg) were administered by tailvein injection, and plasma levels of siRNA were measured periodicallyover 24 hrs by scintillation counting. Cholesterol conjugated siRNA(ALN-3001) was discovered to circulate in mouse plasma for a longerperiod time than unconjugated siRNA (ALN-3000) (FIG. 7). RNAseprotection assays indicated that cholesterol-conjugated siRNA (ALN-3001)was detectable in mouse plasma 12 hours after injection, whereasunconjugated siRNA (ALN-3000) was not detectable in mouse plasma withintwo hours following injection. Similar results were observed in rats.

Mouse liver was harvested at varying time points (ranging from 0.08-24hours) following injection with siRNA, and siRNA localized to the liverwas quantified. Over the time period tested, the amount ofcholesterol-conjugated siRNA (ALN-3001) detected in the liver rangedfrom 14.3-3.55 percent of the total dose administered to the mouse. Theamount of unconjugated siRNA (ALN-3000) detected in the liver was lower,ranging from 3.91-1.75 percent of the total dose administered.

Detection of siRNA in Different Tissues

Various tissues and organs (fat, heart, kidney, liver, and spleen) wereharvested from two CMV-Luc mice 22 hours following injection with 50mg/kg ALN-3001. The antisense strand of the siRNA was detected by RNAseprotection assay. The liver contained the greatest concentration ofsiRNA (˜8-10 μg siRNA/g tissue); the spleen, heart and kidney containedlesser amounts of siRNA (˜2-7 μg siRNA/g tissue); and fat tissuecontained the least amount of siRNA (<˜1 μg siRNA/g tissue).

Glucose-6-Phosphatase siRNA Detection by RNAse Protection Assay

Balbc mice were injected with U/U, 3′C/U, or 3′ C/3′ C siRNA (4 mg/kg)targeting glucose-6-phosphatase (G6Pase) (see Table 18). Administrationwas by hydrodynamic tail vein injection (hd) or non-hydrodynamic tailvein injection (iv), and siRNA was subsequently detected in the liver byRNAse protection assay.

TABLE 18 Test iRNA agents targeting glucose-6-phosphatase siRNADescription U/U No cholesterol; dinucleotide 3′ overhangs on sense andantisense strands 3′C/U dinucleotide 3′ overhangs on sense and antisensestrands; cholesterol conjugated to 3′ end of sense strand(mono-conjugate) 3′C/3′C dinucleotide 3′ overhangs on sense andantisense strands; cholesterol conjugated to 3′ end of both sense andantisense strands (bis-conjugate)

Unconjugated siRNA (U/U) delivered by hd was detected by 15 min.post-injection (the earliest determined time-point) and was stilldetectable in the liver 18 hours post-injection.

Delivery by normal iv administration resulted in the greatestconcentration of 3′C/3′C siRNA (the bis-cholesterol-conjugate) in theliver 1 hour post injection (as compared to themono-cholesterol-conjugate 3′C/3′U siRNA). At 18 hours post injection,3′C/3′C siRNAs and 3′C/U siRNA were still detectable in the liver withthe bis-conjugate at higher levels compared to the mono-conjugate.

While this invention has been particularly shown and described withreference to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-20. (canceled)
 21. An RNA agent for inhibiting the expression of atarget human gene in a cell, comprising a sense sequence and anantisense sequence, wherein the sense sequence has one or moreasymmetrical 2′-O alkyl modifications and the antisense sequence has2-20 phosphorothioate modifications, wherein the antisense sequence hasfewer asymmetrical 2′-O alkyl modifications than the sense sequence,wherein the sense sequence comprises a conjugate group, and wherein theantisense sequence targets the human gene sequence.
 22. The RNA agent ofclaim 21, wherein at least one of the 2′-O-alkyl modifications is a2′-OMe modification.
 23. The RNA agent of claim 21, wherein the sensesequence has 4-12 2′-O-alkyl modifications.
 24. The RNA agent of claim23, wherein at least 4 of the asymmetrical 2′-O-alkyl modifications arewithin the 6 terminal nucleotides of the 5′ end or 3′ end of the sensesequence.
 25. The RNA agent of claim 23, wherein at least 4 of theasymmetrical 2′-O-alkyl modifications are within the 6 terminalnucleotides of the 5′ end or 3′ end of the sense sequence, and at leastone of the 2′-O-alkyl modifications is in another portion of the sensesequence.
 26. The RNA agent of claim 23, wherein at least 4 of theasymmetrical 2′-O-alkyl modifications are within the 4 terminalnucleotides of the 5′ end or 3′ end of the sense sequence.
 27. The RNAagent of claim 21, wherein at least 2 of the phosphorothioatemodifications are within the 2 terminal nucleotides of the 5′ end or 3′end of the antisense sequence.
 28. The RNA agent of claim 27, whereinthe sense sequence further comprises 2 phosphorothioate modificationswithin the 2 terminal nucleotides of the 5′ end or 3′ end of the sensesequence.
 29. The RNA agent of claim 21, wherein the antisense sequencehas at least 3 phosphorothioate modifications.
 30. The RNA agent ofclaim 21, wherein the sense and antisense sequences of the RNA agent arefully complementary to each other.
 31. The RNA agent of claim 21,wherein the RNA agent is at least 21 nucleotides in length, and theduplex region of the RNA agent is about 19 nucleotides in length. 32.The RNA agent of claim 21, wherein the RNA agent has a duplex region ofabout 19-21 nucleotides in length and one or two 3′ overhangs of about 2nucleotides in length.
 33. The RNA agent of claim 21, wherein the sensesequence further comprises at least one asymmetric modification selectedfrom the group consisting of 2′-5′-linkages, L sugars, modified sugars,nucleobase modifications, cation groups, Zwitterionic groups, andconjugate groups.
 34. The RNA agent of claim 33, wherein themodification is 2′-5′ linkages, and the 2′-5′ linkage isphosphorothioate.
 35. The RNA agent of claim 33, wherein themodification is L sugars, and the L sugar is L ribose or L-arabinosesugar.
 36. The RNA agent of claim 33, wherein the modification ismodified sugars, and the modified sugar is a locked nucleic acid, hexosenucleic acid or cyclohexane nucleic acid.
 37. The RNA agent of claim 21,wherein the antisense sequence further comprises at least one asymmetricmodification selected from the group consisting of 2′-5′-linkages, Lsugars, modified sugars, nucleobase modifications, cation groups,Zwitterionic groups, and conjugate groups.
 38. The RNA agent of claim37, wherein the modification is 2′-5′ linkages, and the 2′-5′ linkage isphosphorothioate.
 39. The RNA agent of claim 37, wherein themodification is a L sugar, and the L sugar is L ribose or L-arabinosesugar.
 40. The RNA agent of claim 37, wherein the modification is amodified sugar, and the modified sugar is a locked nucleic acid, ahexose nucleic acid, or a cyclohexane nucleic acid.